Sample records for action site cas

  1. Closure Report for Corrective Action Unit 139: Waste Disposal Sites, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    2009-07-31

    Corrective Action Unit (CAU) 139 is identified in the Federal Facility Agreement and Consent Order (FFACO) as 'Waste Disposal Sites' and consists of the following seven Corrective Action Sites (CASs), located in Areas 3, 4, 6, and 9 of the Nevada Test Site: CAS 03-35-01, Burn Pit; CAS 04-08-02, Waste Disposal Site; CAS 04-99-01, Contaminated Surface Debris; CAS 06-19-02, Waste Disposal Site/Burn Pit; CAS 06-19-03, Waste Disposal Trenches; CAS 09-23-01, Area 9 Gravel Gertie; and CAS 09-34-01, Underground Detection Station. Closure activities were conducted from December 2008 to April 2009 according to the FFACO (1996, as amended February 2008) andmore » the Corrective Action Plan for CAU 139 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office, 2007b). The corrective action alternatives included No Further Action, Clean Closure, and Closure in Place with Administrative Controls. Closure activities are summarized. CAU 139, 'Waste Disposal Sites,' consists of seven CASs in Areas 3, 4, 6, and 9 of the NTS. The closure alternatives included No Further Action, Clean Closure, and Closure in Place with Administrative Controls. This CR provides a summary of completed closure activities, documentation of waste disposal, and confirmation that remediation goals were met. The following site closure activities were performed at CAU 139 as documented in this CR: (1) At CAS 03-35-01, Burn Pit, soil and debris were removed and disposed as LLW, and debris was removed and disposed as sanitary waste. (2) At CAS 04-08-02, Waste Disposal Site, an administrative UR was implemented. No postings or post-closure monitoring are required. (3) At CAS 04-99-01, Contaminated Surface Debris, soil and debris were removed and disposed as LLW, and debris was removed and disposed as sanitary waste. (4) At CAS 06-19-02, Waste Disposal Site/Burn Pit, no work was performed. (5) At CAS 06-19-03, Waste Disposal Trenches, a native soil cover was installed, and a UR was

  2. Closure Report for Corrective Action Unit 104: Area 7 Yucca Flat Atmospheric Test Sites, Nevada National Security Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    None

    2013-06-27

    This Closure Report (CR) presents information supporting closure of Corrective Action Unit (CAU) 104, Area 7 Yucca Flat Atmospheric Test Sites, and provides documentation supporting the completed corrective actions and confirmation that closure objectives for CAU 104 were met. This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; the U.S. Department of Energy (DOE), Environmental Management; the U.S. Department of Defense; and DOE, Legacy Management. CAU 104 consists of the following 15 Corrective Action Sites (CASs), located in Area 7 of the Nevada National Securitymore » Site: · CAS 07-23-03, Atmospheric Test Site T-7C · CAS 07-23-04, Atmospheric Test Site T7-1 · CAS 07-23-05, Atmospheric Test Site · CAS 07-23-06, Atmospheric Test Site T7-5a · CAS 07-23-07, Atmospheric Test Site - Dog (T-S) · CAS 07-23-08, Atmospheric Test Site - Baker (T-S) · CAS 07-23-09, Atmospheric Test Site - Charlie (T-S) · CAS 07-23-10, Atmospheric Test Site - Dixie · CAS 07-23-11, Atmospheric Test Site - Dixie · CAS 07-23-12, Atmospheric Test Site - Charlie (Bus) · CAS 07-23-13, Atmospheric Test Site - Baker (Buster) · CAS 07-23-14, Atmospheric Test Site - Ruth · CAS 07-23-15, Atmospheric Test Site T7-4 · CAS 07-23-16, Atmospheric Test Site B7-b · CAS 07-23-17, Atmospheric Test Site - Climax Closure activities began in October 2012 and were completed in April 2013. Activities were conducted according to the Corrective Action Decision Document/Corrective Action Plan for CAU 104. The corrective actions included No Further Action and Clean Closure. Closure activities generated sanitary waste, mixed waste, and recyclable material. Some wastes exceeded land disposal limits and required treatment prior to disposal. Other wastes met land disposal restrictions and were disposed in appropriate onsite landfills. The U.S. Department of Energy, National Nuclear Security Administration Nevada Field

  3. Corrective Action Plan for Corrective Action Unit 563: Septic Systems, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    This Corrective Action Plan (CAP) has been prepared for Corrective Action Unit (CAU) 563, Septic Systems, in accordance with the Federal Facility Agreement and Consent Order. CAU 563 consists of four Corrective Action Sites (CASs) located in Areas 3 and 12 of the Nevada Test Site. CAU 563 consists of the following CASs: CAS 03-04-02, Area 3 Subdock Septic Tank CAS 03-59-05, Area 3 Subdock Cesspool CAS 12-59-01, Drilling/Welding Shop Septic Tanks CAS 12-60-01, Drilling/Welding Shop Outfalls Site characterization activities were performed in 2007, and the results are presented in Appendix A of the CAU 563 Corrective Action Decision Document.more » The scope of work required to implement the recommended closure alternatives is summarized below. CAS 03-04-02, Area 3 Subdock Septic Tank, contains no contaminants of concern (COCs) above action levels. No further action is required for this site; however, as a best management practice (BMP), all aboveground features (e.g., riser pipes and bumper posts) will be removed, the septic tank will be removed, and all open pipe ends will be sealed with grout. CAS 03-59-05, Area 3 Subdock Cesspool, contains no COCs above action levels. No further action is required for this site; however, as a BMP, all aboveground features (e.g., riser pipes and bumper posts) will be removed, the cesspool will be abandoned by filling it with sand or native soil, and all open pipe ends will be sealed with grout. CAS 12-59-01, Drilling/Welding Shop Septic Tanks, will be clean closed by excavating approximately 4 cubic yards (yd3) of arsenic- and chromium-impacted soil. In addition, as a BMP, the liquid in the South Tank will be removed, the North Tank will be removed or filled with grout and left in place, the South Tank will be filled with grout and left in place, all open pipe ends will be sealed with grout or similar material, approximately 10 yd3 of chlordane-impacted soil will be excavated, and debris within the CAS boundary will be removed

  4. Corrective Action Decision Document/Closure Report for Corrective Action Unit 383: Area E-Tunnel Sites, Nevada Test Site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    This Corrective Action Decision Document/Closure Report (CADD/CR) was prepared by the Defense Threat Reduction Agency (DTRA) for Corrective Action Unit (CAU) 383, Area 12 E-Tunnel Sites, which is the joint responsibility of DTRA and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office (NNSA/NSO). This CADD/CR is consistent with the requirements of the Federal Facility Agreement and Consent Order (FFACO) agreed to by the State of Nevada, the DOE, and the U.S. Department of Defense. Corrective Action Unit 383 is comprised of three Corrective Action Sites (CASs) and two adjacent areas: • CAS 12-06-06, Muckpile •more » CAS 12-25-02, Oil Spill • CAS 12-28-02, Radioactive Material • Drainage below the Muckpile • Ponds 1, 2, and 3 The purpose of this CADD/CR is to provide justification and documentation to support the recommendation for closure with no further corrective action, by placing use restrictions at the three CASs and two adjacent areas of CAU 383.« less

  5. Corrective Action Investigation Plan for Corrective Action Unit 106: Areas 5, 11 Frenchman Flat Atmospheric Sites, Nevada National Security Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patrick Matthews

    2011-07-01

    Corrective Action Unit 106 comprises the four corrective action sites (CASs) listed below: • 05-20-02, Evaporation Pond • 05-23-05, Atmospheric Test Site - Able • 05-45-04, 306 GZ Rad Contaminated Area • 05-45-05, 307 GZ Rad Contaminated Area These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives (CAAs). Additional information will be obtained by conducting a corrective action investigation before evaluating CAAs and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viablemore » CAAs that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the data quality objectives (DQOs) developed on January 19, 2010, by representatives of the Nevada Division of Environmental Protection and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 106. The presence and nature of contamination at CAU 106 will be evaluated based on information collected from a field investigation. The CAU includes land areas impacted by the release of radionuclides from groundwater pumping during the Radionuclide Migration study program (CAS 05-20-02), a weapons-related airdrop test (CAS 05-23-05), and unknown support activities at two sites (CAS 05-45-04 and CAS 05-45-05). The presence and nature of contamination from surface-deposited radiological contamination from CAS 05-23-05, Atmospheric Test Site - Able, and other types of releases (such as migration and excavation as well as any potential releases discovered during the investigation) from the remaining three CASs will be evaluated using soil samples collected from the

  6. Corrective Action Plan for Corrective Action Unit 271: Areas 25, 26, and 27 Septic Systems, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    R. B. Jackson

    2003-05-01

    The Areas 25, 26 and 27 Septic Systems are in the Federal Facility Agreement and Consent Order (FFACO) of 1996 as Corrective Action Unit (CAU) 271. This Corrective Action Plan (CAP) provides selected corrective action alternatives and proposes the closure methodology for CAU 271. CAU 271 is located on the Nevada Test Site (NTS) approximately 105 kilometers (65 miles) northwest of Las Vegas, Nevada, and consists of the following 15 Corrective Action Sites (CAS): CAS 25-04-1, Septic System; CAS 25-04-03, Septic System; CAS25-04-04, Septic System; CAS 25-04-08, Septic System; CAS 25-04-09, Septic System; CAS 25-04-10, Septic System; CAS 25-04-11, Septicmore » System; CAS 26-03-01, Contaminated Water Reservoir; CAS 26-04-1, Septic System; CAS 26-04-02, Septic System; CAS 26-05-01, Radioactive Leachfield; CAS-26-05-03, Septic System; CAS 26-05-04, Septic System; CAS 26-05-05, Septic System; and CAS 27-05-02, Leachfield.« less

  7. Corrective Action Plan for Corrective Action Unit 562: Waste Systems, Nevada National Security Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    2011-04-30

    This Corrective Action Plan has been prepared for Corrective Action Unit (CAU) 562, Waste Systems, in accordance with the Federal Facility Agreement and Consent Order (1996; as amended March 2010). CAU 562 consists of 13 Corrective Action Sites (CASs) located in Areas 2, 23, and 25 of the Nevada National Security Site. Site characterization activities were performed in 2009 and 2010, and the results are presented in Appendix A of the Corrective Action Decision Document for CAU 562. The scope of work required to implement the recommended closure alternatives is summarized. (1) CAS 02-26-11, Lead Shot, will be clean closedmore » by removing shot. (2) CAS 02-44-02, Paint Spills and French Drain, will be clean closed by removing paint and contaminated soil. As a best management practice (BMP), asbestos tile will be removed. (3) CAS 02-59-01, Septic System, will be clean closed by removing septic tank contents. As a BMP, the septic tank will be removed. (4) CAS 02-60-01, Concrete Drain, contains no contaminants of concern (COCs) above action levels. No further action is required; however, as a BMP, the concrete drain will be removed. (5) CAS 02-60-02, French Drain, was clean closed. Corrective actions were completed during corrective action investigation activities. As a BMP, the drain grates and drain pipe will be removed. (6) CAS 02-60-03, Steam Cleaning Drain, will be clean closed by removing contaminated soil. As a BMP, the steam cleaning sump grate and outfall pipe will be removed. (7) CAS 02-60-04, French Drain, was clean closed. Corrective actions were completed during corrective action investigation activities. (8) CAS 02-60-05, French Drain, will be clean closed by removing contaminated soil. (9) CAS 02-60-06, French Drain, contains no COCs above action levels. No further action is required. (10) CAS 02-60-07, French Drain, requires no further action. The french drain identified in historical documentation was not located during corrective action

  8. Corrective action investigation plan for Corrective Action Unit 340, Pesticide Release Sites, Nevada Test Site, Nye County, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    This Correction Action Investigation Plan (CAIP) has been developed in accordance with the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the US Department of Energy, Nevada Operations Office (DOE/NV); the State of Nevada Division of Environmental Protection (NDEP); and the US Department of Defense. As required by the FFACO (1996), this document provides or references all of the specific information for planning investigation activities associated with three Corrective Action Sites (CASs) located at the Nevada Test Site (NTS). These CASs are collectively known as Corrective Action Unit (CAU) 340, Pesticide Release Sites. According to themore » FFACO, CASs are sites that may require corrective action(s) and may include solid waste management units or individual disposal or release sites. These sites are CAS 23-21-01, Area 23 Quonset Hut 800 (Q800) Pesticide Release Ditch; CAS 23-18-03, Area 23 Skid Huts Pesticide Storage; and CAS 15-18-02, Area 15 Quonset Hut 15-11 Pesticide Storage (Q15-11). The purpose of this CAIP for CAU 340 is to direct and guide the investigation for the evaluation of the nature and extent of pesticides, herbicides, and other contaminants of potential concern (COPCs) that were stored, mixed, and/or disposed of at each of the CASs.« less

  9. Corrective Action Investigation Plan for Corrective Action Unit 536: Area 3 Release Site, Nevada Test Site, Nevada (Rev. 0 / June 2003), Including Record of Technical Change No. 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    None

    This Corrective Action Investigation Plan contains the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office's approach to collect the data necessary to evaluate corrective action alternatives (CAAs) appropriate for the closure of Corrective Action Unit (CAU) 536: Area 3 Release Site, Nevada Test Site, Nevada, under the Federal Facility Agreement and Consent Order. Corrective Action Unit 536 consists of a single Corrective Action Site (CAS): 03-44-02, Steam Jenny Discharge. The CAU 536 site is being investigated because existing information on the nature and extent of possible contamination is insufficient to evaluate and recommend corrective action alternatives formore » CAS 03-44-02. The additional information will be obtained by conducting a corrective action investigation (CAI) prior to evaluating CAAs and selecting the appropriate corrective action for this CAS. The results of this field investigation are to be used to support a defensible evaluation of corrective action alternatives in the corrective action decision document. Record of Technical Change No. 1 is dated 3-2004.« less

  10. Corrective Action Plan for Corrective Action Unit 366: Area 11 Plutonium Valley Dispersion Sites, Nevada National Security Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    None

    This Corrective Action Plan has been prepared for Corrective Action Unit (CAU) 366, Area 11 Plutonium Valley Dispersion Sites, in accordance with the Federal Facility Agreement and Consent Order (FFACO, 1996 as amended). CAU 366 consists of the following six Corrective Action Sites (CASs) located in Area 11 of the Nevada National Security Site: · CAS 11-08-01, Contaminated Waste Dump #1 · CAS 11-08-02, Contaminated Waste Dump #2 · CAS 11-23-01, Radioactively Contaminated Area A · CAS 11-23-02, Radioactively Contaminated Area B · CAS 11-23-03, Radioactively Contaminated Area C · CAS 11-23-04, Radioactively Contaminated Area D Site characterization activities weremore » performed in 2011 and 2012, and the results are presented in Appendix A of the Corrective Action Decision Document (CADD) for CAU 366 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2012a). The following closure alternatives were recommended in the CADD: · No further action for CAS 11-23-01 · Closure in place for CASs 11-08-01, 11-08-02, 11-23-02, 11-23-03, and 11-23-04 The scope of work required to implement the recommended closure alternatives includes the following: · Non-engineered soil covers approximately 3 feet thick will be constructed at CAS 11-08-01 over contaminated waste dump (CWD) #1 and at CAS 11-08-02 over CWD #2. · FFACO use restrictions (URs) will be implemented for the areas where the total effective dose (TED) exceeds the final action level (FAL) of 25 millirems per Occasional Use Area year (mrem/OU-yr). The FAL is based on an assumption that the future use of the site includes occasional work activities and that workers will not be assigned to the area on a regular basis. A site worker under this scenario is assumed to be on site for a maximum of 80 hours per year for 5 years. The FFACO UR boundaries will encompass the areas where a worker would be exposed to 25 millirems of radioactivity per year if they are present

  11. CLOSURE REPORT FOR CORRECTIVE ACTION UNIT 204: STORAGE BUNKERS, NEVADA TEST SITE, NEVADA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    Corrective Action Unit (CAU) 330 consists of four Corrective Action Sites (CASs) located in Areas 6, 22, and 23 of the Nevada Test Site (NTS). The unit is listed in the Federal Facility Agreement and Consent Order (FFACO, 1996) as CAU 330: Areas 6, 22, and 23 Tanks and Spill Sites. CAU 330 consists of the following CASs: CAS 06-02-04, Underground Storage Tank (UST) and Piping CAS 22-99-06, Fuel Spill CAS 23-01-02, Large Aboveground Storage Tank (AST) Farm CAS 23-25-05, Asphalt Oil Spill/Tar Release

  12. Closure Report for Corrective Action Unit 563: Septic Systems, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    2010-02-28

    Corrective Action Unit (CAU) 563 is identified in the Federal Facility Agreement and Consent Order (FFACO) as “Septic Systems” and consists of the following four Corrective Action Sites (CASs), located in Areas 3 and 12 of the Nevada Test Site: · CAS 03-04-02, Area 3 Subdock Septic Tank · CAS 03-59-05, Area 3 Subdock Cesspool · CAS 12-59-01, Drilling/Welding Shop Septic Tanks · CAS 12-60-01, Drilling/Welding Shop Outfalls Closure activities were conducted from September to November 2009 in accordance with the FFACO (1996, as amended February 2008) and the Corrective Action Plan for CAU 563. The corrective action alternatives includedmore » No Further Action and Clean Closure.« less

  13. CORRECTIVE ACTION DECISION DOCUMENT FOR CORRECTIVE ACTION UNIT 383: AREA 12 E-TUNNEL SITES, NEVADA TEST SITE, REV. NO. 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mark McLane

    2005-03-01

    This Corrective Action Decision Document (CADD) was prepared by the Defense Threat Reduction Agency (DTRA) and the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office (NNSA/NSO). The recommendations and corrective actions described within this document apply to the future closure of Corrective Action Unit (CAU) 383, Area 12 E-Tunnel Sites, which is a joint DTRA and NNSA/NSO site. The CAU consists of three (3) Corrective Action Sites (CASs): CAS 12-06-06 (Muckpile); CAS 12-25-02 (Oil Spill); and CAS 12-28-02 (Radioactive Material). In addition to these CASs, E-Tunnel Ponds One, Two, and Three, and the Drainage Area above themore » ponds were included since closure of the Muckpile will impact these areas. This CADD is consistent with the requirements of the ''Federal Facility Agreement and Consent Order'' agreed to by the State of Nevada, the U.S. Department of Energy, and the U.S. Department of Defense. The DTRA point of contact is the Nevada Operations Office, Environmental Project Manager; currently Ms. Tiffany A. Lantow. The NNSA/NSO point of contact is the Environmental Restoration, Industrial Sites Project Manager; currently Ms. Janet Appenzeller-Wing. The purpose of this CADD is to identify and provide the rationale for the selection of a recommended corrective action alternative for CAU 383. This document presents the recommended corrective action for CAU 383 (E-Tunnel Sites); however, implementation may be affected by the corrective action (to be determined) for CAU 551 (Area 12 Muckpiles) due to the close proximity of B, C, D, and F-Tunnels. The scope of this CADD consists of the following tasks: (1) Develop corrective action objectives; (2) Identify corrective action alternative screening criteria; (3) Develop corrective action alternatives; (4) Perform detailed and comparative evaluations of the corrective action alternatives in relation to the corrective action objectives and screening criteria; and (5) Recommend and

  14. Corrective Action Decision Document/Closure Report for Corrective Action Unit 274: Septic Systems, Nevada Test Site, Nevada, Rev. No.: 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grant Evenson

    2006-09-01

    This Corrective Action Decision Document/Closure Report has been prepared for Corrective Action Unit 274, Septic Systems, Nevada Test Site (NTS), Nevada in accordance with the ''Federal Facility Agreement and Consent Order'' (1996). Corrective Action Unit (CAU) 274 is comprised of five corrective action sites (CASs): (1) CAS 03-02-01, WX-6 ETS Building Septic System; (2) CAS 06-02-01, Cesspool; (3) CAS 09-01-01, Spill Site; (4) CAS 09-05-01, Leaching Pit; and (5) CAS 20-05-01, Septic System. The purpose of this Corrective Action Decision Document/Closure Report is to provide justification and documentation supporting the closure of CAU 274 with no further corrective action. Tomore » achieve this, corrective action investigation (CAI) activities were performed from November 14 through December 17, 2005 as set forth in the CAU 274 Corrective Action Investigation Plan. The purpose of the CAI was to fulfill the following data needs as defined during the data quality objective (DQO) process: (1) Determine whether contaminants of concern (COCs) are present. (2) If contaminants of concern are present, determine their nature and extent. (3) Provide sufficient information and data to complete appropriate corrective actions. The CAU 274 dataset from the investigation results was evaluated based on the data quality indicator parameters. This evaluation demonstrated the quality and acceptability of the dataset for use in fulfilling the DQO data needs. Analytes detected during the CAI were evaluated against final action levels (FALs) established in this document. No analytes were detected at concentrations exceeding the FALs. No COCs have been released to the soil at CAU 274, and corrective action is not required. Therefore, the DQO data needs were met, and it was determined that no corrective action based on risk to human receptors is necessary for the site. All FALs were calculated using the industrial site worker scenario except for benzo(a)pyrene, which was calculated

  15. Closure Report for Corrective Action Unit 562: Waste Systems, Nevada National Security Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    2012-08-15

    This Closure Report (CR) presents information supporting closure of Corrective Action Unit (CAU) 562, Waste Systems, and provides documentation supporting the completed corrective actions and confirmation that closure objectives for CAU 562 were met. This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; the U.S. Department of Energy (DOE), Environmental Management; the U.S. Department of Defense; and DOE, Legacy Management (FFACO, 1996 as amended). CAU 562 consists of the following 13 Corrective Action Sites (CASs), located in Areas 2, 23, and 25 of the Nevadamore » National Security Site: · CAS 02-26-11, Lead Shot · CAS 02-44-02, Paint Spills and French Drain · CAS 02-59-01, Septic System · CAS 02-60-01, Concrete Drain · CAS 02-60-02, French Drain · CAS 02-60-03, Steam Cleaning Drain · CAS 02-60-04, French Drain · CAS 02-60-05, French Drain · CAS 02-60-06, French Drain · CAS 02-60-07, French Drain · CAS 23-60-01, Mud Trap Drain and Outfall · CAS 23-99-06, Grease Trap · CAS 25-60-04, Building 3123 Outfalls Closure activities began in October 2011 and were completed in April 2012. Activities were conducted according to the Corrective Action Plan for CAU 562 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2011). The corrective actions included No Further Action and Clean Closure. Closure activities generated sanitary waste and hazardous waste. Some wastes exceeded land disposal limits and required offsite treatment prior to disposal. Other wastes met land disposal restrictions and were disposed in appropriate onsite or offsite landfills. NNSA/NSO requests the following: · A Notice of Completion from the Nevada Division of Environmental Protection to NNSA/NSO for closure of CAU 562 · The transfer of CAU 562 from Appendix III to Appendix IV, Closed Corrective Action Units, of the FFACO« less

  16. Corrective Action Plan for Corrective Action Unit 262: Area 25 Septic Systems and Underground Discharge Point, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    K. B. Campbell

    This Corrective Action Plan (CAP) provides selected corrective action alternatives and proposes the closure methodology for Corrective Action Unit (CAU) 262, Area 25 Septic Systems and Underground Discharge Point. CAU 262 is identified in the Federal Facility Agreement and Consent Order (FFACO) of 1996. Remediation of CAU 262 is required under the FFACO. CAU 262 is located in Area 25 of the Nevada Test Site (NTS), approximately 100 kilometers (km) (62 miles [mi]) northwest of Las Vegas, Nevada. The nine Corrective Action Sites (CASs) within CAU 262 are located in the Nuclear Rocket Development Station complex. Individual CASs are locatedmore » in the vicinity of the Reactor Maintenance, Assembly, and Disassembly (R-MAD); Engine Maintenance, Assembly, and Disassembly (E-MAD); and Test Cell C compounds. CAU 262 includes the following CASs as provided in the FFACO (1996); CAS 25-02-06, Underground Storage Tank; CAS 25-04-06, Septic Systems A and B; CAS 25-04-07, Septic System; CAS 25-05-03, Leachfield; CAS 25-05-05, Leachfield; CAS 25-05-06, Leachfield; CAS 25-05-08, Radioactive Leachfield; CAS 25-05-12, Leachfield; and CAS 25-51-01, Dry Well. Figures 2, 3, and 4 show the locations of the R-MAD, the E-MAD, and the Test Cell C CASs, respectively. The facilities within CAU 262 supported nuclear rocket reactor engine testing. Activities associated with the program were performed between 1958 and 1973. However, several other projects used the facilities after 1973. A significant quantity of radioactive and sanitary waste was produced during routine operations. Most of the radioactive waste was managed by disposal in the posted leachfields. Sanitary wastes were disposed in sanitary leachfields. Septic tanks, present at sanitary leachfields (i.e., CAS 25-02-06,2504-06 [Septic Systems A and B], 25-04-07, 25-05-05,25-05-12) allowed solids to settle out of suspension prior to entering the leachfield. Posted leachfields do not contain septic tanks. All CASs located in CAU 262

  17. Corrective Action Investigation Plan for Corrective Action Unit 555: Septic Systems Nevada Test Site, Nevada, Rev. No.: 0 with Errata

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pastor, Laura

    2005-12-01

    This Corrective Action Investigation Plan (CAIP) contains project-specific information including facility descriptions, environmental sample collection objectives, and criteria for conducting site investigation activities at Corrective Action Unit (CAU) 555: Septic Systems, Nevada Test Site (NTS), Nevada. This CAIP has been developed in accordance with the ''Federal Facility Agreement and Consent Order'' (FFACO) (1996) that was agreed to by the State of Nevada, the U.S. Department of Energy (DOE), and the U.S. Department of Defense. Corrective Action Unit 555 is located in Areas 1, 3 and 6 of the NTS, which is approximately 65 miles (mi) northwest of Las Vegas, Nevada,more » and is comprised of the five corrective action sites (CASs) shown on Figure 1-1 and listed below: (1) CAS 01-59-01, Area 1 Camp Septic System; (2) CAS 03-59-03, Core Handling Building Septic System; (3) CAS 06-20-05, Birdwell Dry Well; (4) CAS 06-59-01, Birdwell Septic System; and (5) CAS 06-59-02, National Cementers Septic System. An FFACO modification was approved on December 14, 2005, to include CAS 06-20-05, Birdwell Dry Well, as part of the scope of CAU 555. The work scope was expanded in this document to include the investigation of CAS 06-20-05. The Corrective Action Investigation (CAI) will include field inspections, radiological surveys, geophysical surveys, sampling of environmental media, analysis of samples, and assessment of investigation results, where appropriate. Data will be obtained to support corrective action alternative evaluations and waste management decisions. The CASs in CAU 555 are being investigated because hazardous and/or radioactive constituents may be present in concentrations that could potentially pose a threat to human health and the environment. Existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives for the CASs. Additional information will be generated by conducting a

  18. Closure Report for Corrective Action Unit 573: Alpha Contaminated Sites Nevada National Security Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    This Closure Report (CR) presents information supporting the closure of Corrective Action Unit (CAU) 573: Alpha Contaminated Sites, Nevada National Security Site, Nevada. CAU 573 comprises the two corrective action sites (CASs): 05-23-02-GMX Alpha Contaminated Are-Closure in Place and 05-45-01-Atmospheric Test Site - Hamilton- Clean Closure. The purpose of this CR is to provide justification and documentation supporting the recommendation that no further corrective action is needed for CAU 573 based on the implementation of the corrective actions. Corrective action activities were performed at Hamilton from May 25 through June 30, 2016; and at GMX from May 25 to Octobermore » 27, 2016, as set forth in the Corrective Action Decision Document (CADD)/Corrective Action Plan (CAP) for Corrective Action Unit 573: Alpha Contaminated Sites; and in accordance with the Soils Activity Quality Assurance Plan, which establishes requirements, technical planning, and general quality practices. Verification sample results were evaluated against data quality objective criteria developed by stakeholders that included representatives from the Nevada Division of Environmental Protection and the DOE, National Nuclear Security Administration Nevada Field Office (NNSA/NFO) during the corrective action alternative (CAA) meeting held on November 24, 2015. Radiological doses exceeding the final action level were assumed to be present within the high contamination areas associated with CAS 05-23-02, thus requiring corrective action. It was also assumed that radionuclides were present at levels that require corrective action within the soil/debris pile associated with CAS 05-45-01. During the CAU 573 CAA meeting, the CAA of closure in place with a use restriction (UR) was selected by the stakeholders as the preferred corrective action of the high contamination areas at CAS 05-23-02 (GMX), which contain high levels of removable contamination; and the CAA of clean closure was selected by the

  19. Corrective Action Decision Document for Corrective Action Unit 151: Septic Systems and Discharge Area, Nevada Test Site, Nevada, Rev. No.: 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grant Evenson

    2006-05-01

    This Corrective Action Decision Document has been prepared for Corrective Action Unit (CAU) 151, Septic Systems and Discharge Area, at the Nevada Test Site, Nevada, according to the ''Federal Facility Agreement and Consent Order'' (FFACO) (1996). Corrective Action Unit 151 is comprised of eight corrective action sites (CASs): (1) CAS 02-05-01, UE-2ce Pond; (2) CAS 12-03-01, Sewage Lagoons (6); (3) CAS 12-04-01, Septic Tanks; (4) CAS 12-04-02, Septic Tanks; (5) CAS 12-04-03, Septic Tank; (6) CAS 12-47-01, Wastewater Pond; (7) CAS 18-03-01, Sewage Lagoon; and (8) CAS 18-99-09, Sewer Line (Exposed). The purpose of this Corrective Action Decision Document ismore » to identify and provide the rationale for the recommendation of corrective action alternatives (CAAs) for each of the eight CASs within CAU 151. Corrective action investigation (CAI) activities were performed from September 12 through November 18, 2005, as set forth in the CAU 151 Corrective Action Investigation Plan and Record of Technical Change No. 1. Additional confirmation sampling was performed on December 9, 2005; January 10, 2006; and February 13, 2006. Analytes detected during the CAI were evaluated against appropriate final action levels (FALs) to identify the contaminants of concern for each CAS. The results of the CAI identified contaminants of concern at two of the eight CASs in CAU 151 and required the evaluation of CAAs. Assessment of the data generated from investigation activities conducted at CAU 151 revealed the following: (1) Soils at CASs 02-05-01, 12-04-01, 12-04-02, 12-04-03, 12-47-01, 18-03-01, 18-99-09, and Lagoons B through G of CAS 12-03-01 do not contain contamination at concentrations exceeding the FALs. (2) Lagoon A of CAS 12-03-01 has arsenic above FALs in shallow subsurface soils. (3) One of the two tanks of CAS 12-04-01, System No.1, has polychlorinated biphenyls (aroclor-1254), trichloroethane, and cesium-137 above FALs in the sludge. Both CAS 12-04-01, System No.1 tanks

  20. Corrective Action Investigation Plan for Corrective Action Unit 139: Waste Disposal Sites, Nevada Test Site, Nevada, Rev. No.: 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grant Evenson

    2006-04-01

    Corrective Action Unit (CAU) 139 is located in Areas 3, 4, 6, and 9 of the Nevada Test Site, which is 65 miles northwest of Las Vegas, Nevada. Corrective Action Unit 139 is comprised of the seven corrective action sites (CASs) listed below: (1) 03-35-01, Burn Pit; (2) 04-08-02, Waste Disposal Site; (3) 04-99-01, Contaminated Surface Debris; (4) 06-19-02, Waste Disposal Site/Burn Pit; (5) 06-19-03, Waste Disposal Trenches; (6) 09-23-01, Area 9 Gravel Gertie; and (7) 09-34-01, Underground Detection Station. These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluatemore » and recommend corrective action alternatives with the exception of CASs 09-23-01 and 09-34-01. Regarding these two CASs, CAS 09-23-01 is a gravel gertie where a zero-yield test was conducted with all contamination confined to below ground within the area of the structure, and CAS 09-34-01 is an underground detection station where no contaminants are present. Additional information will be obtained by conducting a corrective action investigation (CAI) before evaluating corrective action alternatives and selecting the appropriate corrective action for the other five CASs where information is insufficient. The results of the field investigation will support a defensible evaluation of viable corrective action alternatives that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the data quality objectives (DQOs) developed on January 4, 2006, by representatives of the Nevada Division of Environmental Protection; U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office; Stoller-Navarro Joint Venture; and Bechtel Nevada. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 139.« less

  1. Corrective Action Decision Document/Corrective Action Plan for Corrective Action Unit 573: Alpha Contaminated Sites Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    CAU 573 comprises the following corrective action sites (CASs): • 05-23-02, GMX Alpha Contaminated Area • 05-45-01, Atmospheric Test Site - Hamilton These two CASs include the release at the Hamilton weapons-related tower test and a series of 29 atmospheric experiments conducted at GMX. The two CASs are located in two distinctly separate areas within Area 5. To facilitate site investigation and data quality objective (DQO) decisions, all identified releases (i.e., CAS components) were organized into study groups. The reporting of investigation results and the evaluation of DQO decisions are at the release level. The corrective action alternatives (CAAs) weremore » evaluated at the FFACO CAS level. The purpose of this CADD/CAP is to evaluate potential CAAs, provide the rationale for the selection of recommended CAAs, and provide the plan for implementation of the recommended CAA for CAU 573. Corrective action investigation (CAI) activities were performed from January 2015 through November 2015, as set forth in the CAU 573 Corrective Action Investigation Plan (CAIP). Analytes detected during the CAI were evaluated against appropriate final action levels (FALs) to identify the contaminants of concern. Assessment of the data generated from investigation activities conducted at CAU 573 revealed the following: • Radiological contamination within CAU 573 does not exceed the FALs (based on the Occasional Use Area exposure scenario). • Chemical contamination within CAU 573 does not exceed the FALs. • Potential source material—including lead plates, lead bricks, and lead-shielded cables—was removed during the investigation and requires no additional corrective action.« less

  2. Closure Report for Corrective Action Unit 536: Area 3 Release Site, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    Corrective Action Unit (CAU) 536 is located in Area 3 of the Nevada Test Site. CAU 536 is listed in the Federal Facility Agreement and Consent Order of 1996 as Area 3 Release Site, and comprises a single Corrective Action Site (CAS): {sm_bullet} CAS 03-44-02, Steam Jenny Discharge The Nevada Division of Environmental Protection (NDEP)-approved corrective action alternative for CAS 03-44-02 is clean closure. Closure activities included removing and disposing of total petroleum hydrocarbon (TPH)- and polyaromatic hydrocarbon (PAH)-impacted soil, soil impacted with plutonium (Pu)-239, and concrete pad debris. CAU 536 was closed in accordance with the NDEP-approved CAU 536more » Corrective Action Plan (CAP), with minor deviations as approved by NDEP. The closure activities specified in the CAP were based on the recommendations presented in the CAU 536 Corrective Action Decision Document (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office, 2004). This Closure Report documents CAU 536 closure activities. During closure activities, approximately 1,000 cubic yards (yd3) of hydrocarbon waste in the form of TPH- and PAH-impacted soil and debris, approximately 8 yd3 of Pu-239-impacted soil, and approximately 100 yd3 of concrete debris were generated, managed, and disposed of appropriately. Additionally, a previously uncharacterized, buried drum was excavated, removed, and disposed of as hydrocarbon waste as a best management practice. Waste minimization techniques, such as the utilization of laboratory analysis to characterize and classify waste streams, were employed during the performance of closure« less

  3. Corrective Action Decision Document/Corrective Action Plan for Corrective Action Unit 547: Miscellaneous Contaminated Waste Sites, Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mark Krauss

    2011-09-01

    The purpose of this CADD/CAP is to present the corrective action alternatives (CAAs) evaluated for CAU 547, provide justification for selection of the recommended alternative, and describe the plan for implementing the selected alternative. Corrective Action Unit 547 consists of the following three corrective action sites (CASs): (1) CAS 02-37-02, Gas Sampling Assembly; (2) CAS 03-99-19, Gas Sampling Assembly; and(3) CAS 09-99-06, Gas Sampling Assembly. The gas sampling assemblies consist of inactive process piping, equipment, and instrumentation that were left in place after completion of underground safety experiments. The purpose of these safety experiments was to confirm that a nuclearmore » explosion would not occur in the case of an accidental detonation of the high-explosive component of the device. The gas sampling assemblies allowed for the direct sampling of the gases and particulates produced by the safety experiments. Corrective Action Site 02-37-02 is located in Area 2 of the Nevada National Security Site (NNSS) and is associated with the Mullet safety experiment conducted in emplacement borehole U2ag on October 17, 1963. Corrective Action Site 03-99-19 is located in Area 3 of the NNSS and is associated with the Tejon safety experiment conducted in emplacement borehole U3cg on May 17, 1963. Corrective Action Site 09-99-06 is located in Area 9 of the NNSS and is associated with the Player safety experiment conducted in emplacement borehole U9cc on August 27, 1964. The CAU 547 CASs were investigated in accordance with the data quality objectives (DQOs) developed by representatives of the Nevada Division of Environmental Protection (NDEP) and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office. The DQO process was used to identify and define the type, amount, and quality of data needed to determine and implement appropriate corrective actions for CAU 547. Existing radiological survey data and historical

  4. Corrective Action Decision Document for Corrective Action Unit 563: Septic Systems, Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grant Evenson

    2008-02-01

    This Corrective Action Decision Document has been prepared for Corrective Action Unit (CAU) 563, Septic Systems, in accordance with the Federal Facility Agreement and Consent Order (FFACO, 1996; as amended January 2007). The corrective action sites (CASs) for CAU 563 are located in Areas 3 and 12 of the Nevada Test Site, Nevada, and are comprised of the following four sites: •03-04-02, Area 3 Subdock Septic Tank •03-59-05, Area 3 Subdock Cesspool •12-59-01, Drilling/Welding Shop Septic Tanks •12-60-01, Drilling/Welding Shop Outfalls The purpose of this Corrective Action Decision Document is to identify and provide the rationale for the recommendation of a correctivemore » action alternative (CAA) for the four CASs within CAU 563. Corrective action investigation (CAI) activities were performed from July 17 through November 19, 2007, as set forth in the CAU 563 Corrective Action Investigation Plan (NNSA/NSO, 2007). Analytes detected during the CAI were evaluated against appropriate final action levels (FALs) to identify the contaminants of concern (COCs) for each CAS. The results of the CAI identified COCs at one of the four CASs in CAU 563 and required the evaluation of CAAs. Assessment of the data generated from investigation activities conducted at CAU 563 revealed the following: •CASs 03-04-02, 03-59-05, and 12-60-01 do not contain contamination at concentrations exceeding the FALs. •CAS 12-59-01 contains arsenic and chromium contamination above FALs in surface and near-surface soils surrounding a stained location within the site. Based on the evaluation of analytical data from the CAI, review of future and current operations at CAS 12-59-01, and the detailed and comparative analysis of the potential CAAs, the following corrective actions are recommended for CAU 563.« less

  5. Closure Report for Corrective Action Unit 151: Septic Systems and Discharge Area, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    2008-04-01

    Corrective Action Unit (CAU) 151 is identified in the Federal Facility Agreement and Consent Order (FFACO) as Septic Systems and Discharge Area. CAU 151 consists of the following eight Corrective Action Sites (CASs), located in Areas 2, 12, and 18 of the Nevada Test Site, approximately 65 miles northwest of Las Vegas, Nevada: (1) CAS 02-05-01, UE-2ce Pond; (2) CAS 12-03-01, Sewage Lagoons (6); (3) CAS 12-04-01, Septic Tanks; (4) CAS 12-04-02, Septic Tanks; (5) CAS 12-04-03, Septic Tank; (6) CAS 12-47-01, Wastewater Pond; (7) CAS 18-03-01, Sewage Lagoon; and (8) CAS 18-99-09, Sewer Line (Exposed). CAU 151 closure activitiesmore » were conducted according to the FFACO (FFACO, 1996; as amended February 2008) and the Corrective Action Plan for CAU 151 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office, 2007) from October 2007 to January 2008. The corrective action alternatives included no further action, clean closure, and closure in place with administrative controls. CAU 151 closure activities are summarized in Table 1. Closure activities generated liquid remediation waste, sanitary waste, hydrocarbon waste, and mixed waste. Waste generated was appropriately managed and disposed. Waste that is currently staged onsite is being appropriately managed and will be disposed under approved waste profiles in permitted landfills. Waste minimization activities included waste characterization sampling and segregation of waste streams. Some waste exceeded land disposal restriction limits and required offsite treatment prior to disposal. Other waste meeting land disposal restrictions was disposed of in appropriate onsite or offsite landfills. Waste disposition documentation is included as Appendix C.« less

  6. Corrective Action Investigation Plan for Corrective Action Unit 127: Areas 25 and 26 Storage Tanks, Nevada Test Site, Nevada (Rev. No.: 0, August 2002)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NNSA /NV

    This Corrective Action Investigation Plan (CAIP) contains the U.S. Department of Energy, National Nuclear Security Administration Nevada Operations Offices's approach to collect the data necessary to evaluate corrective action alternatives appropriate for the closure of Corrective Action Unit (CAU) 127 under the Federal Facility Agreement and Consent Order. Corrective Action Unit 127 is located on the Nevada Test Site approximately 65 miles northwest of Las Vegas, Nevada. This CAU is comprised of 12 Corrective Action Sites (CASs) located at Test Cell C; the Engine Maintenance, Assembly, and Disassembly (E-MAD) Facility; the X-Tunnel in Area 25; the Pluto Disassembly Facility; themore » Pluto Check Station; and the Port Gaston Training Facility in Area 26. These CASs include: CAS 25-01-05, Aboveground Storage Tank (AST); CAS 25-02-02, Underground Storage Tank (UST); CAS 25-23-11, Contaminated Materials; CAS 25-12-01, Boiler; CAS 25-01-06, AST; CAS 25-01-07, AST; CAS 25-02-13, UST; CAS 26- 01-01, Filter Tank (Rad) and Piping; CAS 26-01-02, Filter Tank (Rad); CAS 26-99-01, Radioactively Contaminated Filters; CAS 26-02-01, UST; CAS 26-23-01, Contaminated Liquids Spreader. Based on site history, process knowledge, and previous field efforts, contaminants of potential concern for CAU 127 include radionuclides, metals, total petroleum hydrocarbons, volatile organic compounds, asbestos, and polychlorinated biphenyls. Additionally, beryllium may be present at some locations. The sources of potential releases are varied, but releases of contaminated liquids may have occurred and may have migrated into and impacted soil below and surrounding storage vessels at some of the CASs. Also, at several CASs, asbestos-containing materials may be present on the aboveground structures and may be friable. Exposure pathways are limited to ingestion, inhalation, and dermal contact (adsorption) of soils/sediments or liquids, or inhalation of contaminants by site workers due to disturbance

  7. Corrective Action Decision Document for Corrective Action Unit 204: Storage Bunkers, Nevada Test Site, Nevada: Revision 0, Including Errata Sheet

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office

    2004-04-01

    This Corrective Action Decision Document identifies the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office's corrective action alternative recommendation for each of the corrective action sites (CASs) within Corrective Action Unit (CAU) 204: Storage Bunkers, Nevada Test Site (NTS), Nevada, under the Federal Facility Agreement and Consent Order. An evaluation of analytical data from the corrective action investigation, review of current and future operations at each CAS, and a detailed comparative analysis of potential corrective action alternatives were used to determine the appropriate corrective action for each CAS. There are six CASs in CAU 204, which aremore » all located between Areas 1, 2, 3, and 5 on the NTS. The No Further Action alternative was recommended for CASs 01-34-01, 02-34-01, 03-34-01, and 05-99-02; and a Closure in Place with Administrative Controls recommendation was the preferred corrective action for CASs 05-18-02 and 05-33-01. These alternatives were judged to meet all requirements for the technical components evaluated as well as applicable state and federal regulations for closure of the sites and will eliminate potential future exposure pathways to the contaminated media at CAU 204.« less

  8. CLOSURE REPORT FOR CORRECTIVE ACTION UNIT165: AREA 25 AND 26 DRY WELL AND WASH DOWN AREAS, NEVADA TEST SITE, NEVADA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    BECHTEL NEVADA

    This Closure Report (CR) documents the closure activities for Corrective Action Unit (CAU) 165, Area 25 and 26 Dry Well and Washdown Areas, according to the Federal Facility Agreement and Consent Order (FFACO) of 1996. CAU 165 consists of 8 Corrective Action Sites (CASs) located in Areas 25 and 26 of the Nevada Test Site (NTS). The NTS is located approximately 105 kilometers (65 miles) northwest of Las Vegas, nevada. Site closure activities were performed according to the Nevada Division of Environmental Protection (NDEP)-approved Corrective Action Plan (CAP) for CAU 165. CAU 165 consists of the following CASs: (1) CASmore » 25-07-06, Train Decontamination Area; (2) CAS 25-07-07, Vehicle Washdown; (3) CAS 25-20-01, Lab Drain Dry Well; (4) CAS 25-47-01, Reservoir and French Drain; (5) CAS 25-51-02, Drywell; (6) CAS 25-59-01, Septic System; (7) CAS 26-07-01, Vehicle Washdown Station; and (8) CAS 26-59-01, Septic System. CAU 165, Area 25 and 26 Dry Well and Washdown Areas, consists of eight CASs located in Areas 25 and 26 of the NTS. The approved closure alternatives included No Further Action, Clean Closure, and Closure in Place with Administrative Controls.« less

  9. Corrective Action Decision Document/Closure Report for Corrective Action Unit 560: Septic Systems, Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grant Evenson

    2010-04-01

    of the leach pit chamber and leach rock. The contamination present at CAS 06-05-03 within the leach pit was not feasible to remove. •The surface and subsurface soils within and surrounding the septic system at CAS 06-05-04 contained PCB concentrations above the FAL of 0.74 mg/kg. The lateral and vertical extent of COCs was determined for this CAS. Contaminated soils were removed up to within 18 ft of the building. The remaining contamination is confined to subsurface soils adjacent to and beneath Building CP-162 and was not feasible to remove. •The solid materials within the septic tank and soils immediately surrounding the inlet end of the tank at CAS 06-59-03 contained benzo(a)pyrene above the FAL of 0.21 mg/kg. The soils, tank contents, and tank were removed. Materials remaining at this CAS do not contain contamination exceeding FALs. •The solids contained within the septic tank and inlet pipe at CAS 06-59-05 contained the following contaminants above their respective FALs: PCBs, arsenic, lead, benzo(a)pyrene, and pesticides. The tank and inlet pipe contents were removed. Materials remaining at this CAS do not contain contamination exceeding FALs. Therefore, the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office (NNSA/NSO) provides the following corrective action recommendations: •No further action for CASs 03-51-01, 06-04-02, and 06-59-04, as no contaminants of potential concern were present that exceed FALs. •Closure in place for CAS 06-05-03 under a corrective action with a use restriction (UR) for remaining PCB- and arsenic-impacted potential source material (PSM). The UR form and map have been filed in the NNSA/NSO Facility Information Management System, the FFACO database, and NNSA/NSO CAU/CAS files. •Closure in place for CAS 06-05-04 under a corrective action with a UR for remaining PCBs in soil adjacent to and beneath Building CP-162. The UR form and map have been filed in the NNSA/NSO Facility Information

  10. Addendum to the Closure Report for Corrective Action Unit 355: Area 2 Cellars/Mud Pits Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the November 2003, Closure Report for Corrective Action Unit 355: Area 2 Cellars/Mud Pits as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. The approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modification document, this addendum consists of: • Thismore » cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the cancellation of the URs for: • CAS 02-37-01, Cellar & Mud Pit • CAS 02-37-03, Cellar & Mud Pit • CAS 02-37-04, Cellar & Mud Pit • CAS 02-37-05, Cellar & Mud Pit • CAS 02-37-06, Cellar & Mud Pit • CAS 02-37-07, Cellar & Mud Pit • CAS 02-37-10, Cellar & Mud Pit • CAS 02-37-11, Cellar & Mud Pit • CAS 02-37-12, Cellar & Mud Pit • CAS 02-37-13, Cellar & Mud Pit • CAS 02-37-14, Cellar & Mud Pit • CAS 02-37-15, Cellar & Mud Pit • CAS 02-37-16, Cellar & Mud Pit • CAS 02-37-17, Cellar • CAS 02-37-18, Cellar & Tanks These URs were established as part of Federal Facility Agreement and Consent Order (FFACO) corrective actions and were based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since these URs were established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have

  11. Closure Report for Corrective Action Unit 166: Storage Yards and Contaminated Materials, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    2009-08-01

    Corrective Action Unit (CAU) 166 is identified in the Federal Facility Agreement and Consent Order (FFACO) as 'Storage Yards and Contaminated Materials' and consists of the following seven Corrective Action Sites (CASs), located in Areas 2, 3, 5, and 18 of the Nevada Test Site: CAS 02-42-01, Condo Release Storage Yd - North; CAS 02-42-02, Condo Release Storage Yd - South; CAS 02-99-10, D-38 Storage Area; CAS 03-42-01, Conditional Release Storage Yard; CAS 05-19-02, Contaminated Soil and Drum; CAS 18-01-01, Aboveground Storage Tank; and CAS 18-99-03, Wax Piles/Oil Stain. Closure activities were conducted from March to July 2009 according tomore » the FF ACO (1996, as amended February 2008) and the Corrective Action Plan for CAU 166 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office, 2007b). The corrective action alternatives included No Further Action and Clean Closure. Closure activities are summarized. CAU 166, Storage Yards and Contaminated Materials, consists of seven CASs in Areas 2, 3, 5, and 18 of the NTS. The closure alternatives included No Further Action and Clean Closure. This CR provides a summary of completed closure activities, documentation of waste disposal, and confirmation that remediation goals were met. The following site closure activities were performed at CAU 166 as documented in this CR: (1) At CAS 02-99-10, D-38 Storage Area, approximately 40 gal of lead shot were removed and are currently pending treatment and disposal as MW, and approximately 50 small pieces of DU were removed and disposed as LLW. (2) At CAS 03-42-01, Conditional Release Storage Yard, approximately 7.5 yd{sup 3} of soil impacted with lead and Am-241 were removed and disposed as LLW. As a BMP, approximately 22 ft{sup 3} of asbestos tile were removed from a portable building and disposed as ALLW, approximately 55 gal of oil were drained from accumulators and are currently pending disposal as HW, the portable building was removed and

  12. Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity.

    PubMed

    Lee, Ciaran M; Davis, Timothy H; Bao, Gang

    2018-04-01

    What is the topic of this review? In this review, we analyse the performance of recently described tools for CRISPR/Cas9 guide RNA design, in particular, design tools that predict CRISPR/Cas9 activity. What advances does it highlight? Recently, many tools designed to predict CRISPR/Cas9 activity have been reported. However, the majority of these tools lack experimental validation. Our analyses indicate that these tools have poor predictive power. Our preliminary results suggest that target site accessibility should be considered in order to develop better guide RNA design tools with improved predictive power. The recent adaptation of the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system for targeted genome engineering has led to its widespread application in many fields worldwide. In order to gain a better understanding of the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design, these studies have spawned a plethora of guide RNA (gRNA) design tools with algorithms based solely on direct or indirect sequence features. Here, we demonstrate that the predictive power of these tools is poor, suggesting that sequence features alone cannot accurately inform the cutting efficiency of a particular CRISPR/Cas9 gRNA design. Furthermore, we demonstrate that DNA target site accessibility influences the activity of CRISPR/Cas9. With further optimization, we hypothesize that it will be possible to increase the predictive power of gRNA design tools by including both sequence and target site accessibility metrics. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

  13. Corrective Action Investigation Plan for Corrective Action Unit 568: Area 3 Plutonium Dispersion Sites Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    2014-01-01

    CAU 568 is a grouping of sites where there has been a suspected release of contamination associated with nuclear testing. This document describes the planned investigation of CAU 568, which comprises the following corrective action sites (CASs): • 03-23-17, S-3I Contamination Area • 03-23-19, T-3U Contamination Area • 03-23-20, Otero Contamination Area • 03-23-22, Platypus Contamination Area • 03-23-23, San Juan Contamination Area • 03-23-26, Shrew/Wolverine Contamination Area These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives (CAAs). Additional information will be obtained bymore » conducting a corrective action investigation before evaluating CAAs and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viable CAAs that will be presented in the investigation report.« less

  14. Corrective Action Decision Document for Corrective Action Unit 568. Area 3 Plutonium Dispersion Sites, Nevada National Security Site, Nevada Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    The purpose of this Corrective Action Decision Document is to identify and provide the rationale for the recommendation of corrective action alternatives (CAAs) for the 14 CASs within CAU 568. Corrective action investigation (CAI) activities were performed from April 2014 through May 2015, as set forth in the Corrective Action Investigation Plan for Corrective Action Unit 568: Area 3 Plutonium Dispersion Sites, Nevada National Security Site, Nevada; and in accordance with the Soils Activity Quality Assurance Plan, which establishes requirements, technical planning, and general quality practices. The purpose of the CAI was to fulfill data needs as defined during themore » DQO process. The CAU 568 dataset of investigation results was evaluated based on a data quality assessment. This assessment demonstrated that the dataset is complete and acceptable for use in fulfilling the DQO data needs. Based on the evaluation of analytical data from the CAI, review of future and current operations at the 14 CASs, and the detailed and comparative analysis of the potential CAAs, the following corrective actions are recommended for CAU 568: • No further action is the preferred corrective action for CASs 03-23-17, 03-23-22, 03-23-26. • Closure in place is the preferred corrective action for CAS 03-23-19; 03-45-01; the SE DCBs at CASs 03-23-20, 03-23-23, 03-23-31, 03-23-32, 03-23-33, and 03-23-34; and the Pascal-BHCA at CAS 03-23-31. • Clean closure is the preferred corrective action for CASs 03-08-04, 03-23-30, and 03-26-04; and the four well head covers at CASs 03-23-20, 03-23-23, 03-23-31, and 03-23-33.« less

  15. Corrective Action Decision Document/Closure Report for Corrective Action Unit 482: Area 15 U15a/e Muckpiles and Ponds Nevada Test Site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    This Corrective Action Decision Document /Closure Report (CADD/CR) was prepared by the Defense Threat Reduction Agency (DTRA) for Corrective Action Unit (CAU) 482 U15a/e Muckpiles and Ponds. This CADD/CR is consistent with the requirements of the Federal Facility Agreement and Consent Order agreed to by the State of Nevada, the U.S. Department of Energy, and the U.S. Department of Defense. Corrective Action Unit 482 is comprised of three Corrective Action Sites (CASs) and one adjacent area: CAS 15-06-01, U15e Muckpile; CAS 15-06-02, U15a Muckpile; CAS 15-38-01, Area 15 U15a/e Ponds; and Drainage below the U15a Muckpile. The purpose of thismore » CADD/CR is to provide justification and documentation supporting the recommendation for closure with no further corrective action, by placing use restrictions on the three CASs and the adjacent area of CAU 482. To support this recommendation, a corrective action investigation (CAI) was performed in September 2002. The purpose of the CAI was to fulfill the following data needs as defined during the Data Quality Objective (DQO) process: (1) Determine whether contaminants of concern (COCs) are present. (2) If COCs are present, determine their nature and extent. (3) Provide sufficient information and data to determine appropriate corrective actions. The CAU 482 dataset from the CAI was evaluated based on the data quality indicator parameters. This evaluation demonstrated the quality and acceptability of the dataset for use in fulfilling the DQO data needs. Analytes detected during the CAI were evaluated against final action levels (FALs) established in this document. Tier 2 FALS were determined for the hazardous constituents of total petroleum hydrocarbons (TPH)-diesel-range organics (DRO) and the radionuclides americium (Am)-241, cesium (Cs)-137, plutonium (Pu)-238, and Pu-239. The Tier 2 FALs were calculated for the radionuclides using site-specific information. The hazardous constituents of TPH-DRO were compared to the

  16. Closure Report for Corrective Action Unit 224: Decon Pad and Septic Systems, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    Corrective Action Unit (CAU) 224 is located in Areas 02, 03, 05, 06, 11, and 23 of the Nevada Test Site, which is situated approximately 65 miles northwest of Las Vegas, Nevada. CAU 224 is listed in the Federal Facility Agreement and Consent Order (FFACO) of 1996 as Decon Pad and Septic Systems and is comprised of the following nine Corrective Action Sites (CASs): CAS 02-04-01, Septic Tank (Buried); CAS 03-05-01, Leachfield; CAS 05-04-01, Septic Tanks (4)/Discharge Area; CAS 06-03-01, Sewage Lagoons (3); CAS 06-05-01, Leachfield; CAS 06-17-04, Decon Pad and Wastewater Catch; CAS 06-23-01, Decon Pad Discharge Piping; CASmore » 11-04-01, Sewage Lagoon; and CAS 23-05-02, Leachfield. The Nevada Division of Environmental Protection (NDEP)-approved corrective action alternative for CASs 02-04-01, 03-05-01, 06-03-01, 11-04-01, and 23-05-02 is no further action. As a best management practice, the septic tanks and distribution box were removed from CASs 02-04-01 and 11-04-01 and disposed of as hydrocarbon waste. The NDEP-approved correction action alternative for CASs 05-04-01, 06-05-01, 06-17-04, and 06-23-01 is clean closure. Closure activities for these CASs included removing and disposing of radiologically and pesticide-impacted soil and debris. CAU 224 was closed in accordance with the NDEP-approved CAU 224 Corrective Action Plan (CAP). The closure activities specified in the CAP were based on the recommendations presented in the CAU 224 Corrective Action Decision Document (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office, 2005). This Closure Report documents CAU 224 closure activities. During closure activities, approximately 60 cubic yards (yd3) of mixed waste in the form of soil and debris; approximately 70 yd{sup 3} of sanitary waste in the form of soil, liquid from septic tanks, and concrete debris; approximately 10 yd{sup 3} of hazardous waste in the form of pesticide-impacted soil; approximately 0.5 yd{sup 3} of universal

  17. Corrective Action Investigation Plan for Corrective Action Unit 562: Waste Systems Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alfred Wickline

    2009-04-01

    Corrective Action Unit 562 is located in Areas 2, 23, and 25 of the Nevada Test Site, which is approximately 65 miles northwest of Las Vegas, Nevada. Corrective Action Unit 562 is comprised of the 13 corrective action sites (CASs) listed below: • 02-26-11, Lead Shot • 02-44-02, Paint Spills and French Drain • 02-59-01, Septic System • 02-60-01, Concrete Drain • 02-60-02, French Drain • 02-60-03, Steam Cleaning Drain • 02-60-04, French Drain • 02-60-05, French Drain • 02-60-06, French Drain • 02-60-07, French Drain • 23-60-01, Mud Trap Drain and Outfall • 23-99-06, Grease Trap • 25-60-04, Buildingmore » 3123 Outfalls These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives. Additional information will be obtained by conducting a corrective action investigation before evaluating corrective action alternatives and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viable corrective action alternatives that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the data quality objectives (DQOs) developed on December 11, 2008, by representatives of the Nevada Division of Environmental Protection; U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office; Stoller-Navarro Joint Venture; and National Security Technologies, LLC. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 562. Appendix A provides a detailed discussion of the DQO methodology and the DQOs specific to each CAS. The scope of the corrective action investigation for CAU 562 includes the following activities: • Move surface debris and/or materials, as needed, to facilitate

  18. Corrective Action Decision Document/Closure Report for Corrective Action Unit 375: Area 30 Buggy Unit Craters, Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patrick Matthews

    2011-08-01

    Corrective Action Unit 375 comprises three corrective action sites (CASs): (1) 25-23-22, Contaminated Soils Site; (2) 25-34-06, Test Cell A Bunker; and (3) 30-45-01, U-30a, b, c, d, e Craters. The purpose of this CADD/CR is to provide justification and documentation supporting the recommendation that no further corrective action is needed for CAU 375 based on the implementation of corrective action of closure in place with administrative controls at CAS 25-23-22, no further action at CAS 25-34-06, and closure in place with administrative controls and removal of potential source material (PSM) at CAS 30-45-01. Corrective action investigation (CAI) activities weremore » performed from July 28, 2010, through April 4, 2011, as set forth in the Corrective Action Investigation Plan for Corrective Action Unit 375: Area 30 Buggy Unit Craters. The approach for the CAI was divided into two facets: investigation of the primary release of radionuclides, and investigation of other releases (migration in washes and chemical releases). The purpose of the CAI was to fulfill data needs as defined during the data quality objective (DQO) process. The CAU 375 dataset of investigation results was evaluated based on the data quality assessment. This assessment demonstrated the dataset is acceptable for use in fulfilling the DQO data needs. Investigation results were evaluated against final action levels (FALs) established in this document. A radiological dose FAL of 25 millirem per year was established based on the Remote Work Area exposure scenario (336 hours of annual exposure). Radiological doses exceeding the FAL were assumed to be present within the default contamination boundaries at CASs 25-23-22 and 30-45-01. No contaminants were identified at CAS 25-34-06, and no corrective action is necessary. Potential source material in the form of lead plate, lead-acid batteries, and oil within an abandoned transformer were identified at CAS 30-45-01, and corrective actions were

  19. Addendum to the Closure Report for Corrective Action Unit 394: Areas 12, 18, and 29 Spill/Release Sites Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the September 2003, Closure Report for Corrective Action Unit 394: Areas 12, 18, and 29 Spill/Release Sites as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. The approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modification document, this addendum consistsmore » of: • This cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the cancellation of the URs for: • CAS 12-25-04, UST 12-16-2 Waste Oil Release • CAS 18-25-01, Oil Spills • CAS 18-25-02, Oil Spills • CAS 18-25-03, Oil Spill • CAS 29-44-01, Fuel Spill These URs were established as part of Federal Facility Agreement and Consent Order (FFACO) corrective actions and were based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since these URs were established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, these URs were re-evaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006c). This re-evaluation consisted of comparing the original data (used to define the need for the URs) to risk-based final action levels (FALs) developed using

  20. Corrective Action Investigation Plan for Corrective Action Unit 151: Septic Systems and Discharge Area, Nevada Test Site, Nevada, Rev. No.: 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    David A. Strand

    2004-06-01

    This Corrective Action Investigation Plan (CAIP) contains project-specific information for conducting site investigation activities at Corrective Action Unit (CAU) 151: Septic Systems and Discharge Area, Nevada Test Site, Nevada. Information presented in this CAIP includes facility descriptions, environmental sample collection objectives, and criteria for the selection and evaluation of environmental corrective action alternatives. Corrective Action Unit 151 is located in Areas 2, 12, 18, and 20 of the Nevada Test Site, which is 65 miles northwest of Las Vegas, Nevada. Corrective Action Unit 151 is comprised of the nine Corrective Action Sites (CAS) listed below: (1) 02-05-01, UE-2ce Pond; (2)more » 12-03-01, Sewage Lagoons (6); (3) 12-04-01, Septic Tanks; (4) 12-04-02, Septic Tanks; (5) 12-04-03, Septic Tank; (6) 12-47-01, Wastewater Pond; (7) 18-03-01, Sewage Lagoon; (8) 18-99-09, Sewer Line (Exposed); and (9) 20-19-02, Photochemical Drain. The CASs within CAU 151 are discharge and collection systems. Corrective Action Site 02-05-01 is located in Area 2 and is a well-water collection pond used as a part of the Nash test. Corrective Action Sites 12-03-01, 12-04-01, 12-04-02, 12-04-03, and 12-47-01 are located in Area 12 and are comprised of sewage lagoons, septic tanks, associated piping, and two sumps. The features are a part of the Area 12 Camp housing and administrative septic systems. Corrective Action Sites 18-03-01 and 18-99-09 are located in the Area 17 Camp in Area 18. These sites are sewage lagoons and associated piping. The origin and terminus of CAS 18-99-09 are unknown; however, the type and configuration of the pipe indicates that it may be a part of the septic systems in Area 18. Corrective Action Site 20-19-02 is located in the Area 20 Camp. This site is comprised of a surface discharge of photoprocessing chemicals.« less

  1. Corrective Action Investigation Plan for Corrective Action Unit 576: Miscellaneous Radiological Sites and Debris Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    Corrective Action Unit (CAU) 576 is located in Areas 2, 3, 5, 8, and 9 of the Nevada National Security Site, which is approximately 65 miles northwest of Las Vegas, Nevada. CAU 576 is a grouping of sites where there has been a suspected release of contamination associated with nuclear testing. This document describes the planned investigation of CAU 576, which comprises the following corrective action sites (CASs): 00-99-01, Potential Source Material; 02-99-12, U-2af (Kennebec) Surface Rad-Chem Piping; 03-99-20, Area 3 Subsurface Rad-Chem Piping; 05-19-04, Frenchman Flat Rad Waste Dump ; 09-99-08, U-9x (Allegheny) Subsurface Rad-Chem Piping; 09-99-09, U-9its u24more » (Avens-Alkermes) Surface Contaminated Flex Line These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives (CAAs). Additional information will be obtained by conducting a corrective action investigation before evaluating CAAs and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viable CAAs that will be presented in the Corrective Action Decision Document (CADD).« less

  2. Corrective Action Decision Document for Corrective Action Unit 204: Storage Bunkers, Nevada Test Site, Nevada, Rev. No. 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Robert Boehlecke

    2004-04-01

    The six bunkers included in CAU 204 were primarily used to monitor atmospheric testing or store munitions. The ''Corrective Action Investigation Plan (CAIP) for Corrective Action Unit 204: Storage Bunkers, Nevada Test Site, Nevada'' (NNSA/NV, 2002a) provides information relating to the history, planning, and scope of the investigation; therefore, it will not be repeated in this CADD. This CADD identifies potential corrective action alternatives and provides a rationale for the selection of a recommended corrective action alternative for each CAS within CAU 204. The evaluation of corrective action alternatives is based on process knowledge and the results of investigative activitiesmore » conducted in accordance with the CAIP (NNSA/NV, 2002a) that was approved prior to the start of the Corrective Action Investigation (CAI). Record of Technical Change (ROTC) No. 1 to the CAIP (approval pending) documents changes to the preliminary action levels (PALs) agreed to by the Nevada Division of Environmental Protection (NDEP) and DOE, National Nuclear Security Administration Nevada Site Office (NNSA/NSO). This ROTC specifically discusses the radiological PALs and their application to the findings of the CAU 204 corrective action investigation. The scope of this CADD consists of the following: (1) Develop corrective action objectives; (2) Identify corrective action alternative screening criteria; (3) Develop corrective action alternatives; (4) Perform detailed and comparative evaluations of corrective action alternatives in relation to corrective action objectives and screening criteria; and (5) Recommend and justify a preferred corrective action alternative for each CAS within CAU 204.« less

  3. Closure Report for Corrective Action Unit 574: Neptune, Nevada National Security Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    Corrective Action Unit (CAU) 574 is identified in the Federal Facility Agreement and Consent Order (FFACO) as 'Neptune' and consists of the following two Corrective Action Sites (CASs), located in Area 12 of the Nevada National Security Site: (1) CAS 12-23-10, U12c.03 Crater (Neptune); and (2) CAS 12-45-01, U12e.05 Crater (Blanca). This Closure Report presents information supporting closure of CAU 574 according to the FFACO (FFACO, 1996 [as amended March 2010]) and the Streamlined Approach for Environmental Restoration Plan for CAU 574 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2011). The following activities were performedmore » to support closure of CAU 574: (1) In situ external dose rate measurements were collected using thermoluminescent dosimeters at CAS 12-45-01, U12e.05 Crater (Blanca). (2) Total effective dose rates were determined at both sites by summing the internal and external dose rate components. (3) A use restriction (UR) was implemented at CAS 12-23-10, U12c.03 Crater (Neptune). Areas that exceed the final action level (FAL) of 25 millirems per year (mrem/yr) based on the Occasional Use Area exposure scenario are within the existing use restricted area for CAU 551. The 25-mrem/yr FAL is not exceeded outside the existing CAU 551 UR for any of the exposure scenarios (Industrial Area, Remote Work Area, and Occasional Use Area). Therefore, the existing UR for CAU 551 is sufficient to bound contamination that exceeds the FAL. (4) An administrative UR was implemented at CAS 12-45-01, U12e.05 Crater (Blanca) as a best management practice (BMP). The 25-mrem/yr FAL was not exceeded for the Remote Work Area or Occasional Use Area exposure scenarios; therefore, a UR is not required. However, because the 25-mrem/yr FAL was exceeded for the Industrial Area exposure scenario, an administrative UR was established as a BMP. UR documentation is included as Appendix B. The UR at CAS 12-23-10, U12c.03 Crater

  4. Corrective Action Decision Document for Corrective Action Unit 204: Storage Bunkers, Nevada Test Site, Nevada, Revision 0 with ROTC 1, 2, and Errata

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wickline, Alfred

    2004-04-01

    This Corrective Action Decision Document (CADD) has been prepared for Corrective Action Unit (CAU) 204 Storage Bunkers, Nevada Test Site (NTS), Nevada, in accordance with the ''Federal Facility Agreement and Consent Order'' (FFACO) that was agreed to by the State of Nevada; U.S. Department of Energy (DOE); and the U.S. Department of Defense (FFACO, 1996). The NTS is approximately 65 miles (mi) north of Las Vegas, Nevada (Figure 1-1). The Corrective Action Sites (CASs) within CAU 204 are located in Areas 1, 2, 3, and 5 of the NTS, in Nye County, Nevada (Figure 1-2). Corrective Action Unit 204 ismore » comprised of the six CASs identified in Table 1-1. As shown in Table 1-1, the FFACO describes four of these CASs as bunkers one as chemical exchange storage and one as a blockhouse. Subsequent investigations have identified four of these structures as instrumentation bunkers (CASs 01-34-01, 02-34-01, 03-34-01, 05-33-01), one as an explosives storage bunker (CAS 05-99-02), and one as both (CAS 05-18-02). The six bunkers included in CAU 204 were primarily used to monitor atmospheric testing or store munitions. The ''Corrective Action Investigation Plan (CAIP) for Corrective Action Unit 204: Storage Bunkers, Nevada Test Site, Nevada'' (NNSA/NV, 2002a) provides information relating to the history, planning, and scope of the investigation; therefore, it will not be repeated in this CADD. This CADD identifies potential corrective action alternatives and provides a rationale for the selection of a recommended corrective action alternative for each CAS within CAU 204. The evaluation of corrective action alternatives is based on process knowledge and the results of investigative activities conducted in accordance with the CAIP (NNSA/NV, 2002a) that was approved prior to the start of the Corrective Action Investigation (CAI). Record of Technical Change (ROTC) No. 1 to the CAIP (approval pending) documents changes to the preliminary action levels (PALs) agreed to by the Nevada

  5. Addendum to the Closure Report for Corrective Action Unit 358: Areas 18, 19, 20 Cellars/Mud Pits Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the January 2004, Closure Report for Corrective Action Unit 358: Areas 18, 19, 20 Cellars/Mud Pits as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. The approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modification document, this addendum consists of:more » • This cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the cancellation of the URs for: • CAS 20-23-02, Postshot Cellar • CAS 20-23-03, Cellar • CAS 20-23-04, Postshot Cellar • CAS 20-23-05, Postshot Cellar • CAS 20-23-06, Cellar • CAS 20-37-01, Cellar & Mud Pit • CAS 20-37-05, Cellar These URs were established as part of Federal Facility Agreement and Consent Order (FFACO) corrective actions and were based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since these URs were established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, these URs were re-evaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006c). This re-evaluation consisted of comparing the original data (used to define the need for the URs) to risk

  6. CRISPR-Cas9-Edited Site Sequencing (CRES-Seq): An Efficient and High-Throughput Method for the Selection of CRISPR-Cas9-Edited Clones.

    PubMed

    Veeranagouda, Yaligara; Debono-Lagneaux, Delphine; Fournet, Hamida; Thill, Gilbert; Didier, Michel

    2018-01-16

    The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells). Here we described a next-generation sequencing method called CRISPR-Cas9 Edited Site Sequencing (CRES-Seq) for the efficient and high-throughput screening of CRISPR-Cas9-edited clones. CRES-Seq facilitates the precise genotyping up to 96 CRISPR-Cas9-edited sites (CRES) in a single MiniSeq (Illumina) run with an approximate sequencing cost of $6/clone. CRES-Seq is particularly useful when multiple genes are simultaneously targeted by CRISPR-Cas9, and also for screening of clones generated from multiallelic genes/polyploid cells. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

  7. PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing.

    PubMed

    Malina, Abba; Cameron, Christopher J F; Robert, Francis; Blanchette, Mathieu; Dostie, Josée; Pelletier, Jerry

    2015-12-08

    In CRISPR-Cas9 genome editing, the underlying principles for selecting guide RNA (gRNA) sequences that would ensure for efficient target site modification remain poorly understood. Here we show that target sites harbouring multiple protospacer adjacent motifs (PAMs) are refractory to Cas9-mediated repair in situ. Thus we refine which substrates should be avoided in gRNA design, implicating PAM density as a novel sequence-specific feature that inhibits in vivo Cas9-driven DNA modification.

  8. PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing

    PubMed Central

    Malina, Abba; Cameron, Christopher J. F.; Robert, Francis; Blanchette, Mathieu; Dostie, Josée; Pelletier, Jerry

    2015-01-01

    In CRISPR-Cas9 genome editing, the underlying principles for selecting guide RNA (gRNA) sequences that would ensure for efficient target site modification remain poorly understood. Here we show that target sites harbouring multiple protospacer adjacent motifs (PAMs) are refractory to Cas9-mediated repair in situ. Thus we refine which substrates should be avoided in gRNA design, implicating PAM density as a novel sequence-specific feature that inhibits in vivo Cas9-driven DNA modification. PMID:26644285

  9. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

    PubMed Central

    Isaac, R Stefan; Jiang, Fuguo; Doudna, Jennifer A; Lim, Wendell A; Narlikar, Geeta J; Almeida, Ricardo

    2016-01-01

    The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo. DOI: http://dx.doi.org/10.7554/eLife.13450.001 PMID:27130520

  10. Corrective Action Investigation Plan for Corrective Action Unit 165: Areas 25 and 26 Dry Well and Washdown Areas, Nevada Test Site, Nevada (including Record of Technical Change Nos. 1, 2, and 3) (January 2002, Rev. 0)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    U.S. Department of Energy, National Nuclear Security Administration Nevada Operations Office

    This Corrective Action Investigation Plan contains the U.S. Department of Energy, National Nuclear Security Administration Nevada Operations Office's approach to collect the data necessary to evaluate corrective action alternatives appropriate for the closure of Corrective Action Unit (CAU) 165 under the Federal Facility Agreement and Consent Order. Corrective Action Unit 165 consists of eight Corrective Action Sites (CASs): CAS 25-20-01, Lab Drain Dry Well; CAS 25-51-02, Dry Well; CAS 25-59-01, Septic System; CAS 26-59-01, Septic System; CAS 25-07-06, Train Decontamination Area; CAS 25-07-07, Vehicle Washdown; CAS 26-07-01, Vehicle Washdown Station; and CAS 25-47-01, Reservoir and French Drain. All eight CASsmore » are located in the Nevada Test Site, Nevada. Six of these CASs are located in Area 25 facilities and two CASs are located in Area 26 facilities. The eight CASs at CAU 165 consist of dry wells, septic systems, decontamination pads, and a reservoir. The six CASs in Area 25 are associated with the Nuclear Rocket Development Station that operated from 1958 to 1973. The two CASs in Area 26 are associated with facilities constructed for Project Pluto, a series of nuclear reactor tests conducted between 1961 to 1964 to develop a nuclear-powered ramjet engine. Based on site history, the scope of this plan will be a two-phased approach to investigate the possible presence of hazardous and/or radioactive constituents at concentrations that could potentially pose a threat to human health and the environment. The Phase I analytical program for most CASs will include volatile organic compounds, semivolatile organic compounds, Resource Conservation and Recovery Act metals, total petroleum hydrocarbons, polychlorinated biphenyls, and radionuclides. If laboratory data obtained from the Phase I investigation indicates the presence of contaminants of concern, the process will continue with a Phase II investigation to define the extent of contamination. Based on the

  11. Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy.

    PubMed

    Shibata, Mikihiro; Nishimasu, Hiroshi; Kodera, Noriyuki; Hirano, Seiichi; Ando, Toshio; Uchihashi, Takayuki; Nureki, Osamu

    2017-11-10

    The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9-RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9-RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9.

  12. Corrective Action Investigation Plan for Corrective Action Unit 104: Area 7 Yucca Flat Atmospheric Test Sites, Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patrick Matthews

    2011-08-01

    CAU 104 comprises the 15 CASs listed below: (1) 07-23-03, Atmospheric Test Site T-7C; (2) 07-23-04, Atmospheric Test Site T7-1; (3) 07-23-05, Atmospheric Test Site; (4) 07-23-06, Atmospheric Test Site T7-5a; (5) 07-23-07, Atmospheric Test Site - Dog (T-S); (6) 07-23-08, Atmospheric Test Site - Baker (T-S); (7) 07-23-09, Atmospheric Test Site - Charlie (T-S); (8) 07-23-10, Atmospheric Test Site - Dixie; (9) 07-23-11, Atmospheric Test Site - Dixie; (10) 07-23-12, Atmospheric Test Site - Charlie (Bus); (11) 07-23-13, Atmospheric Test Site - Baker (Buster); (12) 07-23-14, Atmospheric Test Site - Ruth; (13) 07-23-15, Atmospheric Test Site T7-4; (14) 07-23-16,more » Atmospheric Test Site B7-b; (15) 07-23-17, Atmospheric Test Site - Climax These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives (CAAs). Additional information will be obtained by conducting a corrective action investigation before evaluating CAAs and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viable CAAs that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the data quality objectives (DQOs) developed on April 28, 2011, by representatives of the Nevada Division of Environmental Protection and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 104. The releases at CAU 104 consist of surface-deposited radionuclides from 30 atmospheric nuclear tests. The presence and nature of contamination at CAU 104 will be evaluated based on information collected from a field investigation. Radiological contamination will be evaluated based on a

  13. Streamlined Approach for Environmental Restoration (SAFER) Plan for Corrective Action Unit 117: Area 26 Pluto Disassembly Facility, Nevada Test Site, Nevada With Errata Sheets, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pat Matthews

    This Streamlined Approach for Environmental Restoration (SAFER) Plan addresses the actions needed to achieve closure for Corrective Action Unit (CAU) 117, Pluto Disassembly Facility, identified in the Federal Facility Agreement and Consent Order. Corrective Action Unit 117 consists of one Corrective Action Site (CAS), CAS 26-41-01, located in Area 26 of the Nevada Test Site. This plan provides the methodology for field activities needed to gather the necessary information for closing CAS 26-41-01. There is sufficient information and process knowledge from historical documentation and investigations of similar sites regarding the expected nature and extent of potential contaminants to recommend closuremore » of CAU 117 using the SAFER process. Additional information will be obtained by conducting a field investigation before finalizing the appropriate corrective action for this CAS. The results of the field investigation will support a defensible recommendation that no further corrective action is necessary following SAFER activities. This will be presented in a Closure Report that will be prepared and submitted to the Nevada Division of Environmental Protection (NDEP) for review and approval. The site will be investigated to meet the data quality objectives (DQOs) developed on June 27, 2007, by representatives of NDEP; U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office; Stoller-Navarro Joint Venture; and National Security Technologies, LLC. The DQO process was used to identify and define the type, amount, and quality of data needed to determine and implement appropriate corrective actions for CAS 26-41-01 in CAU 117.« less

  14. Corrective Action Decision Document/Closure Report for Corrective Action Unit 219: Septic Systems and Injection Wells, Nevada Test Site, Nevada, Rev. No.: 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    David Strand

    2006-05-01

    This Corrective Action Decision Document/Closure Report has been prepared for Corrective Action Unit (CAU) 219, Septic Systems and Injection Wells, in Areas 3, 16, and 23 of the Nevada Test Site, Nevada, in accordance with the ''Federal Facility Agreement and Consent Order'' (1996). Corrective Action Unit 219 is comprised of the following corrective action sites (CASs): (1) 03-11-01, Steam Pipes and Asbestos Tiles; (2) 16-04-01, Septic Tanks (3); (3) 16-04-02, Distribution Box; (4) 16-04-03, Sewer Pipes; (5) 23-20-01, DNA Motor Pool Sewage and Waste System; and (6) 23-20-02, Injection Well. The purpose of this Corrective Action Decision Document/Closure Report ismore » to provide justification and documentation supporting the recommendation for closure of CAU 219 with no further corrective action beyond the application of a use restriction at CASs 16-04-01, 16-04-02, and 16-04-03. To achieve this, corrective action investigation (CAI) activities were performed from June 20 through October 12, 2005, as set forth in the CAU 219 Corrective Action Investigation Plan and Record of Technical Change No. 1. A best management practice was implemented at CASs 16-04-01, 16-04-02, and 16-04-03, and corrective action was performed at CAS 23-20-01 between January and April 2006. In addition, a use restriction will be applied to CASs 16-04-01, 16-04-02, and 16-04-03 to provide additional protection to Nevada Test Site personnel. The purpose of the CAI was to fulfill the following data needs as defined during the data quality objective (DQO) process: (1) Determine whether contaminants of concern (COCs) are present. (2) If COCs are present, determine their nature and extent. (3) Provide sufficient information and data to complete appropriate corrective actions. The CAU 219 dataset from the investigation results was evaluated based on the data quality indicator parameters. This evaluation demonstrated the quality and acceptability of the dataset for use in fulfilling the DQO data

  15. Corrective Action Decision Document/Closure Report for Corrective Action Unit 374: Area 20 Schooner Unit Crater, Nevada National Security Site, Nevada with ROTC 1 and 2, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    Corrective Action Unit 374 comprises five corrective action sites (CASs): • 18-22-05, Drum • 18-22-06, Drums (20) • 18-22-08, Drum • 18-23-01, Danny Boy Contamination Area • 20-45-03, U-20u Crater (Schooner) The purpose of this Corrective Action Decision Document/Closure Report is to provide justification and documentation supporting the recommendation that no further corrective action is needed for CAU 374 based on the implementation of corrective actions. The corrective action of closure in place with administrative controls was implemented at CASs 18-23-01 and 20-45-03, and a corrective action of removing potential source material (PSM) was conducted at CAS 20-45-03. The othermore » CASs require no further action; however, best management practices of removing PSM and drums at CAS 18-22-06, and removing drums at CAS 18-22-08 were performed. Corrective action investigation (CAI) activities were performed from May 4 through October 6, 2010, as set forth in the Corrective Action Investigation Plan for Corrective Action Unit 374: Area 20 Schooner Unit Crater, Nevada Test Site, Nevada. The approach for the CAI was divided into two facets: investigating the primary release of radionuclides and investigating other releases (migration in washes and chemical releases). The purpose of the CAI was to fulfill data needs as defined during the data quality objective (DQO) process. The CAU 374 dataset of investigation results was evaluated based on the data quality indicator parameters. This evaluation demonstrated the dataset is acceptable for use in fulfilling the DQO data needs. Analytes detected during the CAI were evaluated against final action levels (FALs) established in this document. Radiological doses exceeding the FAL of 25 millirem per year were found to be present in the surface soil that was sampled. It is assumed that radionuclide levels present in subsurface media within the craters and ejecta fields (default contamination boundaries) at the Danny

  16. Corrective Action Decision Document/Closure Report for Corrective Action Unit 365: Baneberry Contamination Area, Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patrick Matthews

    2011-09-01

    Corrective Action Unit 365 comprises one corrective action site (CAS), CAS 08-23-02, U-8d Contamination Area. The purpose of this CADD/CR is to provide justification and documentation supporting the recommendation that no further corrective action is needed for CAU 365 based on the implementation of the corrective action of closure in place with a use restriction (UR). Corrective action investigation (CAI) activities were performed from January 18, 2011, through August 2, 2011, as set forth in the Corrective Action Investigation Plan for Corrective Action Unit 365: Baneberry Contamination Area. The purpose of the CAI was to fulfill data needs as definedmore » during the data quality objective (DQO) process. The CAU 365 dataset of investigation results was evaluated based on a data quality assessment. This assessment demonstrated the dataset is complete and acceptable for use in supporting the DQO decisions. Investigation results were evaluated against final action levels (FALs) established in this document. A radiological dose FAL of 25 millirem per year was established based on the Remote Work Area exposure scenario (336 hours of annual exposure). Radiological doses exceeding the FAL were found to be present to the southwest of the Baneberry crater. It was also assumed that radionuclide levels present within the crater and fissure exceed the FAL. Corrective actions were undertaken that consisted of establishing a UR and posting warning signs for the crater, fissure, and the area located to the southwest of the crater where soil concentrations exceeded the FAL. These URs were recorded in the FFACO database; the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office (NNSA/NSO) Facility Information Management System; and the NNSA/NSO CAU/CAS files. Therefore, NNSA/NSO provides the following recommendations: (1) No further corrective actions beyond what are described in this document are necessary for CAU 365. (2) A Notice of

  17. Corrective Action Decision Document/Closure Report for Corrective Action Unit 477: Area 12 N-Tunnel Muckpile, Nevada Test Site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    This Corrective Action Decision Document (CADD)/Closure Report (CR) was prepared by the Defense Threat Reduction Agency (DTRA) for Corrective Action Unit (CAU) 477, N-Tunnel Muckpile. This CADD/CR is consistent with the requirements of the Federal Facility Agreement and Consent Order (FFACO) agreed to by the State of Nevada, the U.S. Department of Energy, and the U.S. Department of Defense. Corrective Action Unit 477 is comprised of one Corrective Action Site (CAS): • 12-06-03, Muckpile The purpose of this CADD/CR is to provide justification and documentation supporting the recommendation for closure with no further action, by placing use restrictions on CAUmore » 477.« less

  18. Corrective Action Investigation Plan for Corrective Action Unit 563: Septic Systems, Nevada Test Site, Nevada, with Errata Sheet, Revision 0 (in English; Afrikaans)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alfred Wickline

    Corrective Action Unit 563, Septic Systems, is located in Areas 3 and 12 of the Nevada Test Site, which is 65 miles northwest of Las Vegas, Nevada. Corrective Action Unit 563 is comprised of the four corrective action sites (CASs) below: • 03-04-02, Area 3 Subdock Septic Tank • 03-59-05, Area 3 Subdock Cesspool • 12-59-01, Drilling/Welding Shop Septic Tanks • 12-60-01, Drilling/Welding Shop Outfalls These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives. Additional information will be obtained by conducting a corrective actionmore » investigation (CAI) before evaluating corrective action alternatives and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viable corrective action alternatives that will be presented in the Corrective Action Decision Document.« less

  19. Closure Report for Corrective Action Unit 539: Areas 25 and 26 Railroad Tracks Nevada National Security Site, Nevada with ROTC-1, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mark Kauss

    2011-06-01

    This Closure Report (CR) presents information supporting the closure of Corrective Action Unit (CAU) 539: Areas 25 and 26 Railroad Tracks, Nevada National Security Site, Nevada. This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; U.S. Department of Energy (DOE), Environmental Management; U.S. Department of Defense; and DOE, Legacy Management. The corrective action sites (CASs) within CAU 539 are located within Areas 25 and 26 of the Nevada National Security Site. Corrective Action Unit 539 comprises the following CASs: • 25-99-21, Area 25 Railroad Tracksmore » • 26-99-05, Area 26 Railroad Tracks The purpose of this CR is to provide documentation supporting the completed corrective actions and provide data confirming that the closure objectives for CASs within CAU 539 were met. To achieve this, the following actions were performed: • Reviewed documentation on historical and current site conditions, including the concentration and extent of contamination. • Conducted radiological walkover surveys of railroad tracks in both Areas 25 and 26. • Collected ballast and soil samples and calculated internal dose estimates for radiological releases. • Collected in situ thermoluminescent dosimeter measurements and calculated external dose estimates for radiological releases. • Removed lead bricks as potential source material (PSM) and collected verification samples. • Implemented corrective actions as necessary to protect human health and the environment. • Properly disposed of corrective action and investigation wastes. • Implemented an FFACO use restriction (UR) for radiological contamination at CAS 25-99-21. The approved UR form and map are provided in Appendix F and will be filed in the DOE, National Nuclear Security Administration Nevada Site Office (NNSA/NSO), Facility Information Management System; the FFACO database; and the NNSA/NSO CAU/CAS files. From

  20. Corrective Action Decision Document/Closure Report for Corrective Action Unit 478: Area 12 T-Tunnel Ponds, Nevada Test Site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    This Corrective Action Decision Document (CADD)/Closure Report (CR) was prepared by the Defense Threat Reduction Agency (DTRA) for Corrective Action Unit (CAU) 478, Area 12 T-Tunnel Ponds. This CADD/CR is consistent with the requirements of the Federal Facility Agreement and Consent Order (FFACO) agreed to by the State of Nevada, the U.S. Department of Energy (DOE), and the U.S. Department of Defense. Corrective Action Unit 478 is comprised of one corrective action site (CAS): • 12-23-01, Ponds (5) RAD Area The purpose of this CADD/CR is to provide justification and documentation supporting the recommendation for closure in place with usemore » restrictions for CAU 478.« less

  1. Corrective Action Decision Document/Closure Report for Corrective Action Unit 559: T Tunnel Compressor/Blower Pad, Nevada Test Site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    This Corrective Action Decision Document (CADD)/Closure Report (CR) was prepared by the Defense Threat Reduction Agency (DTRA) for Corrective Action Unit (CAU) 559, T-Tunnel Compressor/Blower Pad. This CADD/CR is consistent with the requirements of the Federal Facility Agreement and Consent Order (FFACO) agreed to by the State of Nevada, the U.S. Department of Energy, and the U.S. Department of Defense. Corrective Action Unit 559 is comprised of one Corrective Action Site (CAS): • 12-25-13, Oil Stained Soil and Concrete The purpose of this CADD/CR is to provide justification and documentation supporting the recommendation for closure in place with use restrictionsmore » for CAU 559.« less

  2. Corrective Action Investigation Plan for Corrective Action Unit 374: Area 20 Schooner Unit Crater Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patrick Matthews

    Corrective Action Unit 374 is located in Areas 18 and 20 of the Nevada Test Site, which is approximately 65 miles northwest of Las Vegas, Nevada. Corrective Action Unit 374 comprises the five corrective action sites (CASs) listed below: • 18-22-05, Drum • 18-22-06, Drums (20) • 18-22-08, Drum • 18-23-01, Danny Boy Contamination Area • 20-45-03, U-20u Crater (Schooner) These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives (CAAs). Additional information will be obtained by conducting a corrective action investigation before evaluating CAAsmore » and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viable CAAs that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the data quality objectives (DQOs) developed on October 20, 2009, by representatives of the Nevada Division of Environmental Protection and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 374.« less

  3. Nucleic Acid-Dependent Conformational Changes in CRISPR-Cas9 Revealed by Site-Directed Spin Labeling.

    PubMed

    Vazquez Reyes, Carolina; Tangprasertchai, Narin S; Yogesha, S D; Nguyen, Richard H; Zhang, Xiaojun; Rajan, Rakhi; Qin, Peter Z

    2017-06-01

    In a type II clustered regularly interspaced short palindromic repeats (CRISPR) system, RNAs that are encoded at the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic acid-dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain their function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large-scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR-Cas9, which will provide key information for understanding the mechanism of CRISPR function.

  4. Closure Letter Report for Corrective Action Unit 496: Buried Rocket Site - Antelope Lake

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    A Streamlined Approach for Environmental Restoration (SAFER) Plan for investigation and closure of CAU 496, Corrective Action Site (CAS) TA-55-008-TAAL (Buried Rocket), at the Tonopah Test Range (TTR), was approved by the Nevada Department of Environmental Protection (NDEP) on July 21,2004. Approval to transfer CAS TA-55-008-TAAL from CAU 496 to CAU 4000 (No Further Action Sites) was approved by NDEP on December 21, 2005, based on the assumption that the rocket did not present any environmental concern. The approval letter included the following condition: ''NDEP understands, from the NNSA/NSO letter dated November 30,2005, that a search will be conducted formore » the rocket during the planned characterization of other sites at the Tonopah Test Range and, if found, the rocket will be removed as a housekeeping measure''. NDEP and U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office personnel located the rocket on Mid Lake during a site visit to TTR, and a request to transfer CAS TA-55-008-TAAL from CAU 4000 back to CAU 496 was approved by NDEP on September 11,2006. CAS TA-55-008-TAAL was added to the ''Federal Facility Agreement and Consent Order'' of 1996, based on an interview with a retired TTR worker in 1993. The original interview documented that a rocket was launched from Area 9 to Antelope Lake and was never recovered due to the high frequency of rocket tests being conducted during this timeframe. The interviewee recalled the rocket being an M-55 or N-55 (the M-50 ''Honest John'' rocket was used extensively at TTR from the 1960s to early 1980s). A review of previously conducted interviews with former TTR personnel indicated that the interviewees confused information from several sites. The location of the CAU 496 rocket on Mid Lake is directly south of the TTR rocket launch facility in Area 9 and is consistent with information gathered on the lost rocket during recent interviews. Most pertinently, an interview in 2005 with

  5. Corrective Action Investigation Plan for Corrective Action Unit 371: Johnnie Boy Crater and Pin Stripe Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patrick Matthews

    Corrective Action Unit (CAU) 371 is located in Areas 11 and 18 of the Nevada Test Site, which is approximately 65 miles northwest of Las Vegas, Nevada. Corrective Action Unit 371 is comprised of the two corrective action sites (CASs) listed below: • 11-23-05, Pin Stripe Contamination Area • 18-45-01, U-18j-2 Crater (Johnnie Boy) These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives. Additional information will be obtained by conducting a corrective action investigation before evaluating corrective action alternatives and selecting the appropriate correctivemore » action for each CAS. The results of the field investigation will support a defensible evaluation of viable corrective action alternatives that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the data quality objectives (DQOs) developed on November 19, 2008, by representatives of the Nevada Division of Environmental Protection; U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office; Stoller-Navarro Joint Venture; and National Security Technologies, LLC. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 371. Appendix A provides a detailed discussion of the DQO methodology and the DQOs specific to each CAS. The scope of the corrective action investigation for CAU 371 includes the following activities: • Move surface debris and/or materials, as needed, to facilitate sampling. • Conduct radiological surveys. • Measure in situ external dose rates using thermoluminescent dosimeters or other dose measurement devices. • Collect and submit environmental samples for laboratory analysis to determine internal dose rates. • Combine internal and external dose rates to determine

  6. Corrective Action Plan for Corrective Action Unit 568: Area 3 Plutonium Dispersion Sites Nevada National Security Site, Nevada, Revision 0 with ROTC 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick; Burmeister, Mark

    2016-05-01

    The purpose of this CAP is to provide the plan for implementation of the recommended corrective action alternatives (CAAs) for CAU 568. Site characterization activities were performed in 2014, and the results are presented in Appendix A of the CAU 568 CADD. The CAAs were recommended in the CADD. The scope of work required to implement the recommended CAAs of closure in place and clean closure at 11 of the 14 CASs includes the following: The installation of physical barriers over the nine safety experiment ground zeroes to cover contamination at CASs 03-23-20 (Otero), 03-23-23 (San Juan and Pascal-C), 03-23-31more » (Pascal-B, Luna, Colfax), 03-23-32 (Pascal-A), 03-23-33 (Valencia), and 03-23-34 (Chipmunk); the characterization and removal of three soil and debris piles at CAS 03-08-04, and one HCA soil pile at CAS 03-23-30; the removal of three steel well head covers (PSM) from CASs 03-23-20 (Otero), 03-23-31 (Luna), and 03-23-33 (Valencia); the removal of soil and lead PSM from two locations at CAS 03-26-04; Implementation of FFACO use restrictions at nine safety experiment ground zeroes at CASs 03-23-20, 03-23-23, 03-23-31, 03-23-32, 03-23-33, and 03-23-34; the steel well head cover at CAS 03-23-23; the areas meeting HCA conditions at CASs 03-23-19 and 03-23-31; and the Boomer crater area at CAS 03-45-01. The FFACO use restriction boundaries will be presented in the CAU 568 closure report.« less

  7. Maternal Supply of Cas9 to Zygotes Facilitates the Efficient Generation of Site-Specific Mutant Mouse Models

    PubMed Central

    Cebrian-Serrano, Alberto; Zha, Shijun; Hanssen, Lars; Biggs, Daniel; Preece, Christopher

    2017-01-01

    Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply. PMID:28081254

  8. Corrective Action Investigation Plan for Corrective Action Unit 560: Septic Systems, Nevada Test Site, Nevada with ROTC1, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grant Evenson

    2008-05-01

    Corrective Action Unit (CAU) 560 is located in Areas 3 and 6 of the Nevada Test Site, which is approximately 65 miles northwest of Las Vegas, Nevada. Corrective Action Unit 560 is comprised of the seven corrective action sites (CASs) listed below: • 03-51-01, Leach Pit • 06-04-02, Septic Tank • 06-05-03, Leach Pit • 06-05-04, Leach Bed • 06-59-03, Building CP-400 Septic System • 06-59-04, Office Trailer Complex Sewage Pond • 06-59-05, Control Point Septic System These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend correctivemore » action alternatives. Additional information will be obtained by conducting a corrective action investigation before evaluating corrective action alternatives and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viable corrective action alternatives that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the data quality objectives (DQOs) developed on January 22, 2008, by representatives from the Nevada Division of Environmental Protection; U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office; Stoller-Navarro Joint Venture; and National Security Technologies, LLC. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 560.« less

  9. Streamlined Approach for Environmental Restoration Plan for Corrective Action Unit 574: Neptune, Nevada National Security Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    2011-08-31

    This Streamlined Approach for Environmental Restoration (SAFER) Plan identifies the activities required for closure of Corrective Action Unit (CAU) 574, Neptune. CAU 574 is included in the Federal Facility Agreement and Consent Order (FFACO) (1996 [as amended March 2010]) and consists of the following two Corrective Action Sites (CASs) located in Area 12 of the Nevada National Security Site: (1) CAS 12-23-10, U12c.03 Crater (Neptune); (2) CAS 12-45-01, U12e.05 Crater (Blanca). This plan provides the methodology for the field activities that will be performed to gather the necessary information for closure of the two CASs. There is sufficient information andmore » process knowledge regarding the expected nature and extent of potential contaminants to recommend closure of CAU 574 using the SAFER process. Based on historical documentation, personnel interviews, site process knowledge, site visits, photographs, field screening, analytical results, the results of the data quality objective (DQO) process (Section 3.0), and an evaluation of corrective action alternatives (Appendix B), closure in place with administrative controls is the expected closure strategy for CAU 574. Additional information will be obtained by conducting a field investigation to verify and support the expected closure strategy and provide a defensible recommendation that no further corrective action is necessary. This will be presented in a Closure Report that will be prepared and submitted to the Nevada Division of Environmental Protection (NDEP) for review and approval.« less

  10. CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool.

    PubMed

    Yang, Fayu; Liu, Changbao; Chen, Ding; Tu, Mengjun; Xie, Haihua; Sun, Huihui; Ge, Xianglian; Tang, Lianchao; Li, Jin; Zheng, Jiayong; Song, Zongming; Qu, Jia; Gu, Feng

    2017-06-16

    Cre-loxP, as one of the site-specific genetic manipulation tools, offers a method to study the spatial and temporal regulation of gene expression/inactivation in order to decipher gene function. CRISPR/Cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research. Whether the traditional site-specific genetic manipulation tool and CRISPR/Cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear. Here, we successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time. We also showed that distinct non-homologous end-joining (NHEJ) patterns from CRISPR/Cas9-mediated gene editing of the targeting sequence locates at the level of plasmids (episomal) and chromosomes. Specially, the CRISPR/Cas9-mediated NHEJ pattern in the nuclear genome favors deletions (64%-68% at the human AAVS1 locus versus 4%-28% plasmid DNA). CRISPR/Cas9-loxP, a novel site-specific genetic manipulation tool, offers a platform for the dissection of gene function and molecular insights into DNA-repair pathways. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  11. Addendum to the Closure Report for Corrective Action Unit 356: Mud Pits and Disposal Sites Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the November 2002, Closure Report for Corrective Action Unit 356: Mud Pits and Disposal Sites as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. The approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modification document, this addendum consists of: •more » This cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the cancellation of the URs for: • CAS 03-04-01, Area 3 Change House Septic System • CAS 03-09-04, Mud Pit These URs were established as part of Federal Facility Agreement and Consent Order (FFACO) corrective actions and were based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since these URs were established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, these URs were re-evaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006c). This re-evaluation consisted of comparing the original data (used to define the need for the URs) to risk-based final action levels (FALs) developed using the current Industrial Sites RBCA process. The re-evaluation resulted in a recommendation to remove

  12. How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration.

    PubMed

    Xiao, Yibei; Ng, Sherwin; Nam, Ki Hyun; Ke, Ailong

    2017-10-05

    CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby Cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical. The conserved Cas1 and Cas2 proteins form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer. The prespacer is bound by Cas1-Cas2 as a dual-forked DNA, and the terminal 3'-OH of each 3' overhang serves as an attacking nucleophile during integration. The prespacer is preferentially integrated into the leader-proximal region of the CRISPR array, guided by the leader sequence and a pair of inverted repeats inside the CRISPR repeat. Spacer integration in the well-studied Escherichia coli type I-E CRISPR system also relies on the bacterial integration host factor. In type II-A CRISPR, however, Cas1-Cas2 alone integrates spacers efficiently in vitro; other Cas proteins (such as Cas9 and Csn2) have accessory roles in the biogenesis phase of prespacers. Here we present four structural snapshots from the type II-A system of Enterococcus faecalis Cas1 and Cas2 during spacer integration. Enterococcus faecalis Cas1-Cas2 selectively binds to a splayed 30-base-pair prespacer bearing 4-nucleotide 3' overhangs. Three molecular events take place upon encountering a target: first, the Cas1-Cas2-prespacer complex searches for half-sites stochastically, then it preferentially interacts with the leader-side CRISPR repeat, and finally, it catalyses a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3' overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework to explain the stepwise spacer

  13. Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection.

    PubMed

    Huang, Mengqi; Zhou, Xiaoming; Wang, Huiying; Xing, Da

    2018-02-06

    A novel CRISPR/Cas9 triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase) mediated nucleic acids amplification was developed for rapid and site-specific nucleic acid detection. CAS-EXPAR was primed by the target DNA fragment produced by cleavage of CRISPR/Cas9, and the amplification reaction performed cyclically to generate a large number of DNA replicates which were detected using a real-time fluorescence monitoring method. This strategy that combines the advantages of CRISPR/Cas9 and exponential amplification showed high specificity as well as rapid amplification kinetics. Unlike conventional nucleic acids amplification reactions, CAS-EXPAR does not require exogenous primers, which often cause target-independent amplification. Instead, primers are first generated by Cas9/sgRNA directed site-specific cleavage of target and accumulated during the reaction. It was demonstrated this strategy gave a detection limit of 0.82 amol and showed excellent specificity in discriminating single-base mismatch. Moreover, the applicability of this method to detect DNA methylation and L. monocytogenes total RNA was also verified. Therefore, CAS-EXPAR may provide a new paradigm for efficient nucleic acid amplification and hold the potential for molecular diagnostic applications.

  14. Identification of genomic sites for CRISPR/Cas9-based genome editing in the Vitis vinifera genome.

    PubMed

    Wang, Yi; Liu, Xianju; Ren, Chong; Zhong, Gan-Yuan; Yang, Long; Li, Shaohua; Liang, Zhenchang

    2016-04-21

    CRISPR/Cas9 has been recently demonstrated as an effective and popular genome editing tool for modifying genomes of humans, animals, microorganisms, and plants. Success of such genome editing is highly dependent on the availability of suitable target sites in the genomes to be edited. Many specific target sites for CRISPR/Cas9 have been computationally identified for several annual model and crop species, but such sites have not been reported for perennial, woody fruit species. In this study, we identified and characterized five types of CRISPR/Cas9 target sites in the widely cultivated grape species Vitis vinifera and developed a user-friendly database for editing grape genomes in the future. A total of 35,767,960 potential CRISPR/Cas9 target sites were identified from grape genomes in this study. Among them, 22,597,817 target sites were mapped to specific genomic locations and 7,269,788 were found to be highly specific. Protospacers and PAMs were found to distribute uniformly and abundantly in the grape genomes. They were present in all the structural elements of genes with the coding region having the highest abundance. Five PAM types, TGG, AGG, GGG, CGG and NGG, were observed. With the exception of the NGG type, they were abundantly present in the grape genomes. Synteny analysis of similar genes revealed that the synteny of protospacers matched the synteny of homologous genes. A user-friendly database containing protospacers and detailed information of the sites was developed and is available for public use at the Grape-CRISPR website ( http://biodb.sdau.edu.cn/gc/index.html ). Grape genomes harbour millions of potential CRISPR/Cas9 target sites. These sites are widely distributed among and within chromosomes with predominant abundance in the coding regions of genes. We developed a publicly-accessible Grape-CRISPR database for facilitating the use of the CRISPR/Cas9 system as a genome editing tool for functional studies and molecular breeding of grapes. Among

  15. Corrective Action Decision Document/Closure Report for Corrective Action Unit 561: Waste Disposal Areas, Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mark Krauss

    2011-08-01

    CAU 561 comprises 10 CASs: (1) 01-19-01, Waste Dump; (2) 02-08-02, Waste Dump and Burn Area; (3) 03-19-02, Debris Pile; (4) 05-62-01, Radioactive Gravel Pile; (5) 12-23-09, Radioactive Waste Dump; (6) 22-19-06, Buried Waste Disposal Site; (7) 23-21-04, Waste Disposal Trenches ; (8) 25-08-02, Waste Dump; (9) 25-23-21, Radioactive Waste Dump; and (10) 25-25-19, Hydrocarbon Stains and Trench. The purpose of this CADD/CR is to provide justification and documentation supporting the recommendation for closure of CAU 561 with no further corrective action. The purpose of the CAI was to fulfill the following data needs as defined during the DQO process:more » (1) Determine whether COCs are present; (2) If COCs are present, determine their nature and extent; and (3) Provide sufficient information and data to complete appropriate corrective actions. The following contaminants were determined to be present at concentrations exceeding their corresponding FALs: (1) No contamination exceeding FALs was identified at CASs 01-19-01, 03-19-02, 05-62-01, 12-23-09, and 22-19-06. (2) The surface and subsurface soil within the burn area at CAS 02-08-02 contains arsenic and lead above the FALs of 23 milligrams per kilogram (mg/kg) and 800 mg/kg, respectively. The surface and subsurface soil within the burn area also contains melted lead slag (potential source material [PSM]). The soil within the waste piles contains polyaromatic hydrocarbons (PAHs) above the FALs. The contamination within the burn area is spread throughout the area, as it was not feasible to remove all the PSM (melted lead), while at the waste piles, the contamination is confined to the piles. (3) The surface and subsurface soils within Trenches 3 and 5 at CAS 23-21-04 contain arsenic and polychlorinated biphenyls (PCBs) above the FALs of 23 mg/kg and 0.74 mg/kg, respectively. The soil was removed from both trenches, and the soil that remains at this CAS does not contain contamination exceeding the FALs. Lead bricks and

  16. Closure Report for Corrective Action Unit 117: Area 26 Pluto Disassembly Facility, Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mark Burmeister

    This Closure Report (CR) presents information supporting the closure of Corrective Action Unit (CAU) 117: Area 26 Pluto Disassembly Facility, Nevada Test Site, Nevada. This CR complies with the requirements of the Federal Facility Agreement and Consent Order that was agreed to by the State of Nevada; U.S. Department of Energy (DOE), Environmental Management; U.S. Department of Defense; and DOE, Legacy Management. Corrective Action Unit 117 comprises Corrective Action Site (CAS) 26-41-01, Pluto Disassembly Facility, located in Area 26 of the Nevada Test Site. The purpose of this CR is to provide documentation supporting the completed corrective actions and providemore » data confirming that the closure objectives for CAU 117 were met. To achieve this, the following actions were performed: • Review the current site conditions, including the concentration and extent of contamination. • Implement any corrective actions necessary to protect human health and the environment. • Properly dispose of corrective action and investigation wastes. • Document Notice of Completion and closure of CAU 117 issued by the Nevada Division of Environmental Protection. From May 2008 through February 2009, closure activities were performed as set forth in the Streamlined Approach for Environmental Restoration Plan for Corrective Action Unit 117, Area 26 Pluto Disassembly Facility, Nevada Test Site, Nevada. The purpose of the activities as defined during the data quality objectives process were: • Determine whether contaminants of concern (COCs) are present. • If COCs are present, determine their nature and extent, implement appropriate corrective actions, and properly dispose of wastes. Analytes detected during the closure activities were evaluated against final action levels to determine COCs for CAU 117. Assessment of the data generated from closure activities indicated that the final action levels were exceeded for polychlorinated biphenyls (PCBs) reported as total Aroclor

  17. A machine learning approach for predicting CRISPR-Cas9 cleavage efficiencies and patterns underlying its mechanism of action.

    PubMed

    Abadi, Shiran; Yan, Winston X; Amar, David; Mayrose, Itay

    2017-10-01

    The adaptation of the CRISPR-Cas9 system as a genome editing technique has generated much excitement in recent years owing to its ability to manipulate targeted genes and genomic regions that are complementary to a programmed single guide RNA (sgRNA). However, the efficacy of a specific sgRNA is not uniquely defined by exact sequence homology to the target site, thus unintended off-targets might additionally be cleaved. Current methods for sgRNA design are mainly concerned with predicting off-targets for a given sgRNA using basic sequence features and employ elementary rules for ranking possible sgRNAs. Here, we introduce CRISTA (CRISPR Target Assessment), a novel algorithm within the machine learning framework that determines the propensity of a genomic site to be cleaved by a given sgRNA. We show that the predictions made with CRISTA are more accurate than other available methodologies. We further demonstrate that the occurrence of bulges is not a rare phenomenon and should be accounted for in the prediction process. Beyond predicting cleavage efficiencies, the learning process provides inferences regarding patterns that underlie the mechanism of action of the CRISPR-Cas9 system. We discover that attributes that describe the spatial structure and rigidity of the entire genomic site as well as those surrounding the PAM region are a major component of the prediction capabilities.

  18. How Type II CRISPR-Cas establish immunity through Cas1-Cas2 mediated spacer integration

    PubMed Central

    Xiao, Yibei; Ng, Sherwin; Nam, Ki Hyun; Ke, Ailong

    2017-01-01

    CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the nearby cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes1–5. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer6–9. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical7–9. The conserved Cas1 and Cas2 proteins form an integrase complex consisting two distal Cas1 dimers bridged by a Cas2 dimer in the middle6,10. The prespacer is bound by Cas1-Cas2 as a dual forked DNA, and the terminal 3′-OH of each 3′-overhang serves as an attacking nucleophile during integration11–14. Importantly, the prespacer is preferentially integrated into the leader-proximal region of the CRISPR array1,7,10,15, guided by the leader sequence and a pair of inverted repeats (IRs) inside the CRISPR repeat7,15–20. Spacer integration in the most well-studied Escherichia coli Type I-E CRISPR system further relies on the bacterial Integration Host Factor (IHF)21,22. In Type II-A CRISPR, however, Cas1-Cas2 alone integrates spacer efficiently in vitro18; other Cas proteins (Cas9 and Csn2) play accessory roles in prespacer biogenesis17,23. Focusing on the Enterococcus faecalis Type II-A system24, here we report four structure snapshots of Cas1-Cas2 during spacer integration. EfaCas1-Cas2 selectively binds to a splayed 30-bp prespacer bearing 4-nt 3′-overhangs. Three molecular events take place upon encountering a target: Cas1-Cas2/prespacer first searches for half-sites stochastically, then preferentially interacts with the leader-side CRISPR repeat and catalyzes a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3′-overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework explaining

  19. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

    PubMed

    Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

    2014-05-06

    In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

  20. Corrective Action Investigation Plan for Corrective Action Unit 541: Small Boy Nevada National Security Site and Nevada Test and Training Range, Nevada with ROTC 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    Corrective Action Unit (CAU) 541 is co-located on the boundary of Area 5 of the Nevada National Security Site and Range 65C of the Nevada Test and Training Range, approximately 65 miles northwest of Las Vegas, Nevada. CAU 541 is a grouping of sites where there has been a suspected release of contamination associated with nuclear testing. This document describes the planned investigation of CAU 541, which comprises the following corrective action sites (CASs): 05-23-04, Atmospheric Tests (6) - BFa Site; 05-45-03, Atmospheric Test Site - Small Boy. These sites are being investigated because existing information on the nature andmore » extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives (CAAs). Additional information will be obtained by conducting a corrective action investigation before evaluating CAAs and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viable CAAs that will be presented in the investigation report. The sites will be investigated based on the data quality objectives (DQOs) developed on April 1, 2014, by representatives of the Nevada Division of Environmental Protection; U.S. Air Force; and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Field Office. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 541. The site investigation process also will be conducted in accordance with the Soils Activity Quality Assurance Plan, which establishes requirements, technical planning, and general quality practices to be applied to this activity. The potential contamination sources associated with CASs 05-23-04 and 05-45-03 are from nuclear testing activities conducted at the Atmospheric Tests (6) - BFa Site and Atmospheric Test Site - Small Boy sites. The presence and nature of

  1. Corrective Action Decision Document/Closure Report for Corrective Action Unit 367: Area 10 Sedan, Ess and Uncle Unit Craters Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patrick Matthews

    2011-06-01

    Corrective Action Unit 367 comprises four corrective action sites (CASs): • 10-09-03, Mud Pit • 10-45-01, U-10h Crater (Sedan) • 10-45-02, Ess Crater Site • 10-45-03, Uncle Crater Site The purpose of this Corrective Action Decision Document/Closure Report is to provide justification and documentation of the corrective actions and site closure activities implemented at CAU 367. A corrective action of closure in place with use restrictions was completed at each of the three crater CASs (10-45-01, 10-45-02, and 10-45-03); corrective actions were not required at CAS 10-09-03. In addition, a limited soil removal corrective action was conducted at the locationmore » of a potential source material release. Based on completion of these correction actions, no additional corrective action is required at CAU 367, and site closure is considered complete. Corrective action investigation (CAI) activities were performed from February 2010 through March 2011, as set forth in the Corrective Action Investigation Plan for Corrective Action Unit 367: Area 10 Sedan, Ess and Uncle Unit Craters, Nevada Test Site, Nevada. The approach for the CAI was divided into two facets: investigation of the primary release of radionuclides, and investigation of non-test or other releases (e.g., migration in washes and potential source material). Based on the proximity of the Uncle, Ess, and Sedan craters, the impact of the Sedan test on the fallout deposited from the two earlier tests, and aerial radiological surveys, the CAU 367 investigation was designed to study the releases from the three crater CASs as one combined release (primary release). Corrective Action Site 10-09-03, Mud Pit, consists of two mud pits identified at CAU 367. The mud pits are considered non-test releases or other releases and were investigated independent of the three crater CASs. The purpose of the CAI was to fulfill data needs as defined during the data quality objective (DQO) process. The CAU 367 dataset of

  2. Addendum to the Closure Report for Corrective Action Unit 322: Areas 1 & 3 Release Sites and Injection Wells Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the June 2006, Closure Report for Corrective Action Unit 322: Areas 1 & 3 Release Sites and Injection Wells as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. The approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modification document, thismore » addendum consists of: • This cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the cancellation of the URs for: • CAS 01-25-01, AST Release • CAS 03-25-03, Mud Plant AST Diesel Release These URs were established as part of Federal Facility Agreement and Consent Order (FFACO) corrective actions and were based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since these URs were established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, these URs were re-evaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006c). This re-evaluation consisted of comparing the original data (used to define the need for the URs) to risk-based final action levels (FALs) developed using the current Industrial Sites RBCA process. The re-evaluation resulted in a

  3. Potential pitfalls of CRISPR/Cas9-mediated genome editing.

    PubMed

    Peng, Rongxue; Lin, Guigao; Li, Jinming

    2016-04-01

    Recently, a novel technique named the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)9 system has been rapidly developed. This genome editing tool has improved our ability tremendously with respect to exploring the pathogenesis of diseases and correcting disease mutations, as well as phenotypes. With a short guide RNA, Cas9 can be precisely directed to target sites, and functions as an endonuclease to efficiently produce breaks in DNA double strands. Over the past 30 years, CRISPR has evolved from the 'curious sequences of unknown biological function' into a promising genome editing tool. As a result of the incessant development in the CRISPR/Cas9 system, Cas9 co-expressed with custom guide RNAs has been successfully used in a variety of cells and organisms. This genome editing technology can also be applied to synthetic biology, functional genomic screening, transcriptional modulation and gene therapy. However, although CRISPR/Cas9 has a broad range of action in science, there are several aspects that affect its efficiency and specificity, including Cas9 activity, target site selection and short guide RNA design, delivery methods, off-target effects and the incidence of homology-directed repair. In the present review, we highlight the factors that affect the utilization of CRISPR/Cas9, as well as possible strategies for handling any problems. Addressing these issues will allow us to take better advantage of this technique. In addition, we also review the history and rapid development of the CRISPR/Cas system from the time of its initial discovery in 2012. © 2015 FEBS.

  4. Corrective Action Investigation Plan for Corrective Action Unit 219: Septic Systems and Injection Wells, Nevada Test Site, Nevada, Rev. No.: 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    David A. Strand

    The Corrective Action Investigation Plan for Corrective Action Unit 219, Septic Systems and Injection Wells, has been developed in accordance with the ''Federal Facility Agreement and Consent Order'' (1996) that was agreed to by the State of Nevada, the U.S. Department of Energy, and the U.S. Department of Defense. The purpose of the investigation is to ensure that adequate data are collected to provide sufficient and reliable information to identify, evaluate, and select technically viable corrective actions. Corrective Action Unit 219 is located in Areas 3, 16, and 23 of the Nevada Test Site, which is 65 miles northwest ofmore » Las Vegas, Nevada. Corrective Action Unit 219 is comprised of the six Corrective Action Sites (CASs) listed below: (1) 03-11-01, Steam Pipes and Asbestos Tiles; (2) 16-04-01, Septic Tanks (3); (3) 16-04-02, Distribution Box; (4) 16-04-03, Sewer Pipes; (5) 23-20-01, DNA Motor Pool Sewage and Waste System; and (6) 23-20-02, Injection Well. These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives. Additional information will be obtained by conducting a corrective action investigation prior to evaluating corrective action alternatives and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viable corrective action alternatives that will be presented in the Corrective Action Decision Document.« less

  5. [Clustered regularly interspaced short palindromic repeat associated protein genes cas1 and cas2 in Shigella].

    PubMed

    Xue, Zerun; Wang, Yingfang; Duan, Guangcai; Wang, Pengfei; Wang, Linlin; Guo, Xiangjiao; Xi, Yuanlin

    2014-05-01

    To detect the distribution of clustered regularly interspaced short palindromic repeat (CRISPR) associated protein genes cas1 and cas2 in Shigella and to understand the characteristics of CRISPR with relationship between CRISPR and related characteristics on drug resistance. CRISPR associated protein genes cas1 and cas2 in Shigella were detected by PCR, with its products sequenced and compared. The CRISPR-associated protein genes cas1 and cas2 were found in all the 196 Shigella isolates which were isolated at different times and locations in China. Consistencies showed through related sequencing appeared as follows: cas2, cas1 (a) and cas1 (b) were 96.44%, 97.61% and 96.97%, respectively. There were two mutations including 3177129 site(C→G)and 3177126 site (G→C) of cas1 (b) gene in 2003135 strain which were not found in the corresponding sites of Z23 and 2008113. showed that in terms of both susceptibility and antibiotic-resistance, strain 2003135 was stronger than Z23 and 2008113. CRISPR system widely existed in Shigella, with the level of drug resistance in cas1 (b) gene mutant strains higher than in wild strains. Cas1 (b) gene mutation might be one of the reasons causing the different levels of resistance.

  6. The action of Escherichia coli CRISPR–Cas system on lytic bacteriophages with different lifestyles and development strategies

    PubMed Central

    Strotskaya, Alexandra; Savitskaya, Ekaterina; Metlitskaya, Anastasia; Morozova, Natalia; Datsenko, Kirill A.; Semenova, Ekaterina

    2017-01-01

    Abstract CRISPR–Cas systems provide prokaryotes with adaptive defense against bacteriophage infections. Given an enormous variety of strategies used by phages to overcome their hosts, one can expect that the efficiency of protective action of CRISPR–Cas systems against different viruses should vary. Here, we created a collection of Escherichia coli strains with type I-E CRISPR–Cas system targeting various positions in the genomes of bacteriophages λ, T5, T7, T4 and R1-37 and investigated the ability of these strains to resist the infection and acquire additional CRISPR spacers from the infecting phage. We find that the efficiency of CRISPR–Cas targeting by the host is determined by phage life style, the positions of the targeted protospacer within the genome, and the state of phage DNA. The results also suggest that during infection by lytic phages that are susceptible to CRISPR interference, CRISPR–Cas does not act as a true immunity system that saves the infected cell but rather enforces an abortive infection pathway leading to infected cell death with no phage progeny release. PMID:28130424

  7. Closure Report for Corrective Action Unit 516: Septic Systems and Discharge Points

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    Corrective Action Unit (CAU) 516 is located in Areas 3, 6, and 22 of the Nevada Test Site. CAU 516 is listed in the Federal Facility Agreement and Consent Order of 1996 as Septic Systems and Discharge Points, and is comprised of six Corrective Action Sites (CASs): {sm_bullet} CAS 03-59-01, Bldg 3C-36 Septic System {sm_bullet} CAS 03-59-02, Bldg 3C-45 Septic System {sm_bullet} CAS 06-51-01, Sump and Piping {sm_bullet} CAS 06-51-02, Clay Pipe and Debris {sm_bullet} CAS 06-51-03, Clean Out Box and Piping {sm_bullet} CAS 22-19-04, Vehicle Decontamination Area The Nevada Division of Environmental Protection (NDEP)-approved corrective action alternative for CASsmore » 06-51-02 and 22-19-04 is no further action. The NDEP-approved corrective action alternative for CASs 03-59-01, 03-59-02, 06-51-01, and 06-51-03 is clean closure. Closure activities included removing and disposing of total petroleum hydrocarbon (TPH)-impacted septic tank contents, septic tanks, distribution/clean out boxes, and piping. CAU 516 was closed in accordance with the NDEP-approved CAU 516 Corrective Action Plan (CAP). The closure activities specified in the CAP were based on the recommendations presented in the CAU 516 Corrective Action Decision Document (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office, 2004). This Closure Report documents CAU 516 closure activities. During closure activities, approximately 186 tons of hydrocarbon waste in the form of TPH-impacted soil and debris, as well as 89 tons of construction debris, were generated and managed and disposed of appropriately. Waste minimization techniques, such as field screening of soil samples and the utilization of laboratory analysis to characterize and classify waste streams, were employed during the performance of closure work.« less

  8. The action of Escherichia coli CRISPR-Cas system on lytic bacteriophages with different lifestyles and development strategies.

    PubMed

    Strotskaya, Alexandra; Savitskaya, Ekaterina; Metlitskaya, Anastasia; Morozova, Natalia; Datsenko, Kirill A; Semenova, Ekaterina; Severinov, Konstantin

    2017-02-28

    CRISPR-Cas systems provide prokaryotes with adaptive defense against bacteriophage infections. Given an enormous variety of strategies used by phages to overcome their hosts, one can expect that the efficiency of protective action of CRISPR-Cas systems against different viruses should vary. Here, we created a collection of Escherichia coli strains with type I-E CRISPR-Cas system targeting various positions in the genomes of bacteriophages λ, T5, T7, T4 and R1-37 and investigated the ability of these strains to resist the infection and acquire additional CRISPR spacers from the infecting phage. We find that the efficiency of CRISPR-Cas targeting by the host is determined by phage life style, the positions of the targeted protospacer within the genome, and the state of phage DNA. The results also suggest that during infection by lytic phages that are susceptible to CRISPR interference, CRISPR-Cas does not act as a true immunity system that saves the infected cell but rather enforces an abortive infection pathway leading to infected cell death with no phage progeny release. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Corrective Action Decision Document/Closure Report for Corrective Action Unit 106: Area 5, 11 Frenchman Flat Atmospheric Sites, Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patrick Matthews and Dawn Peterson

    2011-09-01

    Corrective Action Unit 106 comprises four corrective action sites (CASs): (1) 05-20-02, Evaporation Pond; (2) 05-23-05, Atmospheric Test Site - Able; (3) 05-45-04, 306 GZ Rad Contaminated Area; (4) 05-45-05, 307 GZ Rad Contaminated Area. The purpose of this CADD/CR is to provide justification and documentation supporting the recommendation that no further corrective action is needed for CAU 106 based on the implementation of corrective actions. The corrective action of clean closure was implemented at CASs 05-45-04 and 05-45-05, while no corrective action was necessary at CASs 05-20-02 and 05-23-05. Corrective action investigation (CAI) activities were performed from October 20,more » 2010, through June 1, 2011, as set forth in the Corrective Action Investigation Plan for Corrective Action Unit 106: Areas 5, 11 Frenchman Flat Atmospheric Sites. The approach for the CAI was divided into two facets: investigation of the primary release of radionuclides, and investigation of other releases (mechanical displacement and chemical releases). The purpose of the CAI was to fulfill data needs as defined during the data quality objective (DQO) process. The CAU 106 dataset of investigation results was evaluated based on a data quality assessment. This assessment demonstrated the dataset is complete and acceptable for use in fulfilling the DQO data needs. Investigation results were evaluated against final action levels (FALs) established in this document. A radiological dose FAL of 25 millirem per year was established based on the Industrial Area exposure scenario (2,250 hours of annual exposure). The only radiological dose exceeding the FAL was at CAS 05-45-05 and was associated with potential source material (PSM). It is also assumed that additional PSM in the form of depleted uranium (DU) and DU-contaminated debris at CASs 05-45-04 and 05-45-05 exceed the FAL. Therefore, corrective actions were undertaken at these CASs that consisted of removing PSM and collecting

  10. Corrective Action Investigation Plan for Corrective Action Unit 224: Decon Pad and Septic Systems Nevada Test Site, Nevada, Rev. No.: 0, with ROTC 1 and 2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    David A. Strand

    This Corrective Action Investigation Plan (CAIP) contains project-specific information including facility descriptions, environmental sample collection objectives, and criteria for conducting site investigation activities at Corrective Action Unit (CAU) 224: Decon Pad and Septic Systems, Nevada Test Site (NTS), Nevada. This CAIP has been developed in accordance with the ''Federal Facility Agreement and Consent Order'' (FFACO) (1996) that was agreed to by the State of Nevada, the U.S. Department of Energy (DOE), and the U.S. Department of Defense (DoD). The NTS is approximately 65 miles (mi) northwest of Las Vegas, Nevada (Figure 1-1). Corrective Action Unit 224 is comprised of themore » nine Corrective Action Sites (CASs) listed below: 02-04-01, Septic Tank (Buried); 03-05-01, Leachfield; 05-04-01, Septic Tanks (4)/Discharge Area; 06-03-01, Sewage Lagoons (3); 06-05-01, Leachfield; 06-17-04, Decon Pad and Wastewater Catch; 06-23-01, Decon Pad Discharge Piping; 11-04-01, Sewage Lagoon; and 23-05-02, Leachfield. Corrective Action Sites 06-05-01, 06-23-01, and 23-05-02 were identified in the 1991 Reynolds Electrical & Engineering Co., Inc. (REECo) inventory (1991). The remaining sites were identified during review of various historical documents. Additional information will be obtained by conducting a corrective action investigation (CAI) prior to evaluating and selecting a corrective action alternative for each CAS. The CAI will include field inspections, radiological and geological surveys, and sample collection. Data will also be obtained to support investigation-derived waste (IDW) disposal and potential future waste management decisions.« less

  11. Streamlined Approach for Environmental Restoration (SAFER) Plan for Corrective Action Unit 544: Cellars, Mud Pits, and Oil Spills, Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mark Krauss

    2010-07-01

    This Streamlined Approach for Environmental Restoration (SAFER) Plan addresses the actions needed to achieve closure for Corrective Action Unit (CAU) 544, Cellars, Mud Pits, and Oil Spills, identified in the Federal Facility Agreement and Consent Order (FFACO). Corrective Action Unit 544 comprises the following 20 corrective action sites (CASs) located in Areas 2, 7, 9, 10, 12, 19, and 20 of the Nevada Test Site (NTS): • 02-37-08, Cellar & Mud Pit • 02-37-09, Cellar & Mud Pit • 07-09-01, Mud Pit • 09-09-46, U-9itsx20 PS #1A Mud Pit • 10-09-01, Mud Pit • 12-09-03, Mud Pit • 19-09-01, Mudmore » Pits (2) • 19-09-03, Mud Pit • 19-09-04, Mud Pit • 19-25-01, Oil Spill • 19-99-06, Waste Spill • 20-09-01, Mud Pits (2) • 20-09-02, Mud Pit • 20-09-03, Mud Pit • 20-09-04, Mud Pits (2) • 20-09-06, Mud Pit • 20-09-07, Mud Pit • 20-09-10, Mud Pit • 20-25-04, Oil Spills • 20-25-05, Oil Spills This plan provides the methodology for field activities needed to gather the necessary information for closing each CAS. There is sufficient information and process knowledge from historical documentation and investigations of similar sites regarding the expected nature and extent of potential contaminants to recommend closure of CAU 544 using the SAFER process. Using the approach approved for previous mud pit investigations (CAUs 530–535), 14 mud pits have been identified that • are either a single mud pit or a system of mud pits, • are not located in a radiologically posted area, and • have no evident biasing factors based on visual inspections. These 14 mud pits are recommended for no further action (NFA), and further field investigations will not be conducted. For the sites that do not meet the previously approved closure criteria, additional information will be obtained by conducting a field investigation before selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible

  12. Closure Report for Corrective Action Unit 261: Area 25 Test Cell A Leachfield System, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    T. M. Fitzmaurice

    2001-04-01

    The purpose of this Closure Report (CR) is to provide documentation of the completed corrective action at the Test Cell A Leachfield System and to provide data confirming the corrective action. The Test Cell A Leachfield System is identified in the Federal Facility Agreement and Consent Order (FFACO) of 1996 as Corrective Action Unit (CAU) 261. Remediation of CAU 261 is required under the FFACO (1996). CAU 261 is located in Area 25 of the Nevada Test Site (NTS) which is approximately 140 kilometers (87 miles) northwest of Las Vegas, Nevada (Figure 1). CAU 261 consists of two Corrective Actionmore » Sites (CASS): CAS 25-05-01, Leachfield; and CAS 25-05-07, Acid Waste Leach Pit (AWLP) (Figures 2 and 3). Test Cell A was operated during the 1960s and 1970s to support the Nuclear Rocket Development Station. Various operations within Building 3124 at Test Cell A resulted in liquid waste releases to the Leachfield and the AWLP. The following existing site conditions were reported in the Corrective Action Decision Document (CADD) (U.S. Department of Energy, Nevada Operations Office [DOE/NV], 1999): Soil in the leachfield was found to exceed the Nevada Division of Environmental Protection (NDEP) Action Level for petroleum hydrocarbons, the U.S. Environmental Protection Agency (EPA) preliminary remediation goals for semi volatile organic compounds, and background concentrations for strontium-90; Soil below the sewer pipe and approximately 4.5 meters (m) (15 feet [ft]) downstream of the initial outfall was found to exceed background concentrations for cesium-137 and strontium-90; Sludge in the leachfield septic tank was found to exceed the NDEP Action Level for petroleum hydrocarbons and to contain americium-241, cesium-137, uranium-234, uranium-238, potassium-40, and strontium-90; No constituents of concern (COC) were identified at the AWLP. The NDEP-approved CADD (DOWNV, 1999) recommended Corrective Action Alternative 2, ''Closure of the Septic Tank and Distribution Box

  13. Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish.

    PubMed

    Hu, Peinan; Zhao, Xueying; Zhang, Qinghua; Li, Weiming; Zu, Yao

    2018-03-02

    The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites ( tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system. Copyright © 2018 Hu et al.

  14. Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish

    PubMed Central

    Hu, Peinan; Zhao, Xueying; Zhang, Qinghua; Li, Weiming; Zu, Yao

    2018-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites (tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system. PMID:29295818

  15. Corrective Action Decision Document for Corrective Action Unit 340: Pesticide Release sites, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    DOE /NV

    This Corrective Action Decision Document has been prepared for Corrective Action Unit 340, the NTS Pesticide Release Sites, in accordance with the Federal Facility Agreement and Consent Order of 1996 (FFACO, 1996). Corrective Action Unit 340 is located at the Nevada Test Site, Nevada, and is comprised of the following Corrective Action Sites: 23-21-01, Area 23 Quonset Hut 800 Pesticide Release Ditch; 23-18-03, Area 23 Skid Huts Pesticide Storage; and 15-18-02, Area 15 Quonset Hut 15-11 Pesticide Storage. The purpose of this Corrective Action Decision Document is to identify and provide a rationale for the selection of a recommended correctivemore » action alternative for each Corrective Action Site. The scope of this Corrective Action Decision Document consists of the following tasks: Develop corrective action objectives; Identify corrective action alternative screening criteria; Develop corrective action alternatives; Perform detailed and comparative evaluations of the corrective action alternatives in relation to the corrective action objectives and screening criteria; and Recommend and justify a preferred corrective action alternative for each Corrective Action Site.« less

  16. A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium.

    PubMed

    Palanisamy, Arun P; Suryakumar, Geetha; Panneerselvam, Kavin; Willey, Christopher D; Kuppuswamy, Dhandapani

    2015-12-01

    Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src's adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either β-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24-48 h PO myocardium. Our studies indicate that c-Src's adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium. © 2015 Wiley Periodicals, Inc.

  17. A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium

    PubMed Central

    Palanisamy, Arun P.; Suryakumar, Geetha; Panneerselvam, Kavin; Willey, Christopher D.; Kuppuswamy, Dhandapani

    2017-01-01

    Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src’s adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either β-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24–48 h PO myocardium. Our studies indicate that c-Src’s adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium. PMID:25976166

  18. CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites

    PubMed Central

    Naito, Yuki; Hino, Kimihiro; Bono, Hidemasa; Ui-Tei, Kumiko

    2015-01-01

    Summary: CRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a ∼20 nt targeted sequence. The target sequence requirements are twofold. First, the 5′-NGG protospacer adjacent motif (PAM) sequence must be located adjacent to the target sequence. Second, the target sequence should be specific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users to easily select rational target sequences with minimized off-target sites by performing exhaustive searches against genomic sequences. The server currently incorporates the genomic sequences of human, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast. Availability: Freely available at http://crispr.dbcls.jp/. Contact: y-naito@dbcls.rois.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25414360

  19. Addendum to the Closure Report for Corrective Action Unit 214: Bunkers and Storage Areas Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the September 2006, Closure Report for Corrective Action Unit 214: Bunkers and Storage Areas as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. The approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modification document, this addendum consists of: • Thismore » cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the cancellation of the URs for: • CAS 25-23-01, Contaminated Materials • CAS 25-23-19, Radioactive Material Storage These URs were established as part of Federal Facility Agreement and Consent Order (FFACO) corrective actions and were based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since these URs were established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, these URs were re-evaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006c). This re-evaluation consisted of comparing the original data (used to define the need for the URs) to risk-based final action levels (FALs) developed using the current Industrial Sites RBCA process. The re-evaluation resulted in a recommendation

  20. Post-Closure Monitoring Report for Corrective Action Unit 339: Area 12 Fleet Operations Steam Cleaning Effluent Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    K. B. Campbell

    2002-09-01

    The Area 12 Fleet Operations Steam Cleaning Effluent site is located in the southeastern portion of the Area 12 Camp at the Nevada Test Site. This site is identified in the Federal Facility Agreement and Consent Order (1996) as Corrective Action Site (CAS) 12-19-01 and is the only CAS assigned to Corrective Action Unit (CAU) 339. Post-closure sampling and inspection of the site were completed on March 27, 2002. Post-closure monitoring activities were scheduled biennially (every two years) in the Post-Closure Monitoring Plan provided in the Closure Report for CAU 339: Area 12 Fleet Operations Steam Cleaning Effluent, Nevada Testmore » Site (U.S. Department of Energy, Nevada Operations Office [DOEN], 1997). A baseline for the site was established by sampling in 1997. Based on the recommendations from the 1999 post-closure monitoring report (DOE/NV, 1999), samples were collected in 2000, earlier than originally proposed, because the 1999 sample results did not provide the expected decrease in total petroleum hydrocarbon (TPH) concentrations at the site. Sampling results from 2000 (DOE/NV, 2000) and 2001 (DOE/NV, 2001) revealed favorable conditions for natural degradation at the CAU 339 site, but because of differing sample methods and heterogeneity of the soil, data results from 2000 and later were not directly correlated with previous results. Post-closure monitoring activities for 2002 consisted of the following: (1) Soil sample collection from three undisturbed plots (Plots A, B, and C, Figure 2). (2) Sample analysis for TPH as oil and bio-characterization parameters (Comparative Enumeration Assay [CEA] and Standard Nutrient Panel [SNP]). (3) Site inspection to evaluate the condition of the fencing and signs. (4) Preparation and submittal of the Post-Closure Monitoring Report.« less

  1. CRISPR-Cas adaptation: insights into the mechanism of action.

    PubMed

    Amitai, Gil; Sorek, Rotem

    2016-02-01

    Since the first demonstration that CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against phages and plasmids, numerous studies have yielded key insights into the molecular mechanisms governing how these systems attack and degrade foreign DNA. However, the molecular mechanisms underlying the adaptation stage, in which new immunological memory is formed, have until recently represented a major unresolved question. In this Progress article, we discuss recent discoveries that have shown both how foreign DNA is identified by the CRISPR-Cas adaptation machinery and the molecular basis for its integration into the chromosome to form an immunological memory. Furthermore, we describe the roles of each of the specific CRISPR-Cas components that are involved in memory formation, and consider current models for their evolutionary origin.

  2. Addendum to the Closure Report for Corrective Action Unit 326: Areas 6 and 27 Release Sites, Nevada Test Site, Nevada, Revision 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grant Evenson

    This document constitutes an addendum to the Closure Report for Corrective Action Unit 326: Areas 6 and 27 Release Sites, Nevada Test Site, Nevada (Revision 1), December 2002 as described in the document Supplemental Investigation Report for FFACO Use Restrictions, Nevada Test Site, Nevada (SIR) dated November 2008. The SIR document was approved by NDEP on December 5, 2008. The approval of the SIR document constituted approval of each of the recommended UR removals. In conformance with the SIR document, this addendum consists of: • This page that refers the reader to the SIR document for additional information • Themore » cover, title, and signature pages of the SIR document • The NDEP approval letter • The corresponding section of the SIR document This addendum provides the documentation justifying the cancellation of the UR for CAS 06-25-01, CP-1 Heating Oil Release. This UR was established as part of a Federal Facility Agreement and Consent Order (FFACO) corrective action and is based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996). Since this UR was established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, this UR was reevaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006). This re-evaluation consisted of comparing the original data (used to define the need for the UR) to risk-based final action levels (FALs) developed using the current Industrial Sites RBCA process. The re-evaluation resulted in a recommendation to remove the UR because contamination is not present at the site above the risk-based FALs. Requirements for inspecting and maintaining this UR will be canceled, and the postings and signage at this site will be removed. Fencing and posting may be present at this site that are

  3. The Assessment of a Tutoring Program to Meet CAS Standards Using a SWOT Analysis and Action Plan

    ERIC Educational Resources Information Center

    Fullmer, Patricia

    2009-01-01

    This article summarizes the use of SWOT (Strengths, Weaknesses, Opportunities, and Threats) analysis and subsequent action planning as a tool of self-assessment to meet CAS (Council for the Advancement of Standards in Higher Education) requirements for systematic assessment. The use of the evaluation results to devise improvements to increase the…

  4. Corrective Action Investigation Plan for Corrective Action Unit 527: Horn Silver Mine, Nevada Test Site, Nevada: Revision 1 (Including Records of Technical Change No.1, 2, 3, and 4)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    U.S. Department of Energy, National Nuclear Security Administration Nevada Operations Office

    This Corrective Action Investigation Plan contains the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Operations Office's approach to collect the data necessary to evaluate corrective action alternatives appropriate for the closure of Corrective Action Unit (CAU) 527, Horn Silver Mine, Nevada Test Site, Nevada, under the Federal Facility Agreement and Consent Order. Corrective Action Unit 527 consists of one Corrective Action Site (CAS): 26-20-01, Contaminated Waste Dump No.1. The site is located in an abandoned mine site in Area 26 (which is the most arid part of the NTS) approximately 65 miles northwest of Las Vegas. Historicalmore » documents may refer to this site as CAU 168, CWD-1, the Wingfield mine (or shaft), and the Wahmonie mine (or shaft). Historical documentation indicates that between 1959 and the 1970s, nonliquid classified material and unclassified waste was placed in the Horn Silver Mine's shaft. Some of the waste is known to be radioactive. Documentation indicates that the waste is present from 150 feet to the bottom of the mine (500 ft below ground surface). This CAU is being investigated because hazardous constituents migrating from materials and/or wastes disposed of in the Horn Silver Mine may pose a threat to human health and the environment as well as to assess the potential impacts associated with any potential releases from the waste. The results of this field investigation will support a defensible evaluation of corrective action alternatives in the corrective action decision document.« less

  5. Addendum to the Closure Report for Corrective Action Unit 398: Area 25 Spill Sites, Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the April 2003, Closure Report for Corrective Action Unit 398: Area 25 Spill Sites as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. The approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modification document, this addendum consists of: • Thismore » cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the cancellation of the UR for CAS 25-25-17, Subsurface Hydraulic Oil Spill. This UR was established as part of a Federal Facility Agreement and Consent Order (FFACO) corrective action and is based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since this UR was established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, this UR was re-evaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006c). This re-evaluation consisted of comparing the original data (used to define the need for the UR) to risk-based final action levels (FALs) developed using the current Industrial Sites RBCA process. The re-evaluation resulted in a recommendation to remove the UR because contamination is not present at the site

  6. Corrective Action Decision Document/Closure Report for Corrective Action Unit 372: Area 20 Cabriolet/Palanquin Unit Craters, Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick and Sloop, Christy

    2011-04-01

    contamination areas and within the craters at Palanquin and Cabriolet exceed the FAL. It is also assumed that potential source material in the form of lead bricks at Little Feller I and lead-acid batteries at Palanquin and Cabriolet exceed the FAL. Therefore, corrective actions were undertaken that consist of removing potential source material, where present, and implementing a use restriction and posting warning signs at each CAS. These use restrictions were recorded in the FFACO database; the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office (NNSA/NSO) Facility Information Management System; and the NNSA/NSO CAU/CAS files. Therefore, NNSA/NSO provides the following recommendations: • No further corrective actions are necessary for CAU 372. • A Notice of Completion to NNSA/NSO is requested from the Nevada Division of Environmental Protection for closure of CAU 372. • Corrective Action Unit 372 should be moved from Appendix III to Appendix IV of the FFACO.« less

  7. Structure and Engineering of Francisella novicida Cas9

    PubMed Central

    Hirano, Hisato; Gootenberg, Jonathan S.; Horii, Takuro; Abudayyeh, Omar O.; Kimura, Mika; Hsu, Patrick D.; Nakane, Takanori; Ishitani, Ryuichiro; Hatada, Izuho; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

    2016-01-01

    Summary The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA, and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5′-NGG-3′ PAM, and used the structural information to create a variant that can recognize the more relaxed 5′-YG-3′ PAM. Furthermore, we demonstrated that pre-assembled FnCas9 ribonucleoprotein complexes can be microinjected into mouse zygotes to edit endogenous sites with the 5′-YG-3′ PAMs, thus expanding the target space of the CRISPR-Cas9 toolbox. PMID:26875867

  8. Structure and Engineering of Francisella novicida Cas9.

    PubMed

    Hirano, Hisato; Gootenberg, Jonathan S; Horii, Takuro; Abudayyeh, Omar O; Kimura, Mika; Hsu, Patrick D; Nakane, Takanori; Ishitani, Ryuichiro; Hatada, Izuho; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

    2016-02-25

    The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. CRISPR-Cas9 Structures and Mechanisms.

    PubMed

    Jiang, Fuguo; Doudna, Jennifer A

    2017-05-22

    Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

  10. Corrective Action Investigation Plan for Corrective Action Unit 375: Area 30 Buggy Unit Craters, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patrick Matthews

    2010-03-01

    a Plowshare test where five nuclear devices were buried 140 feet (ft) deep in a row at 150-ft intervals. These devices were detonated on March 12, 1968, to produce a trench 254 ft wide, 865 ft long, and 70 ft deep. The mesa where the test was conducted is surrounded on three sides by ravines, and the entire end of the mesa is fenced and posted as a contamination area. These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend CAAs. Additional information will be obtained by conducting a corrective action investigation before evaluating CAAs and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viable CAAs that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the data quality objectives (DQOs) developed on December 2, 2009, by representatives of the Nevada Division of Environmental Protection and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 375.« less

  11. Addendum to the Closure Report for Corrective Action Unit 335: Area 6 Injection Well and Drain Pit Nevada Test Site, Nevada, Revison 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the June 2003, Closure Report for Corrective Action Unit 335: Area 6 Injection Well and Drain Pit as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. The approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modification document, this addendum consistsmore » of: • This cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the cancellation of the URs for: • CAS 06-20-02, 20-inch Cased Hole • CAS 06-23-03, Drain Pit These URs were established as part of Federal Facility Agreement and Consent Order (FFACO) corrective actions and were based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since these URs were established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, these URs were re-evaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006c). This re-evaluation consisted of comparing the original data (used to define the need for the URs) to risk-based final action levels (FALs) developed using the current Industrial Sites RBCA process. The re-evaluation resulted in a recommendation to remove these URs

  12. Exploiting CRISPR/Cas systems for biotechnology

    PubMed Central

    Sampson, Timothy R.; Weiss, David S.

    2015-01-01

    The Cas9 endonuclease is the central component of the Type II CRISPR/Cas system, a prokaryotic adaptive restriction system against invading nucleic acids, such as those originating from bacteriophages and plasmids. Recently, this RNA-directed DNA endonuclease has been harnessed to target DNA sequences of interest. Here, we review the development of Cas9 as an important tool to not only edit the genomes of a number of different prokaryotic and eukaryotic species, but also as an efficient system for site-specific transcriptional repression or activation. Additionally, a specific Cas9 protein has been observed to target an RNA substrate, suggesting that Cas9 may have the ability to be programmed to target RNA as well. Cas proteins from other CRISPR/Cas subtypes may also be exploited in this regard. Thus, CRISPR/Cas systems represent an effective and versatile biotechnological tool, which will have significant impact on future advancements in genome engineering. PMID:24323919

  13. Exploiting CRISPR/Cas systems for biotechnology.

    PubMed

    Sampson, Timothy R; Weiss, David S

    2014-01-01

    The Cas9 endonuclease is the central component of the Type II CRISPR/Cas system, a prokaryotic adaptive restriction system against invading nucleic acids, such as those originating from bacteriophages and plasmids. Recently, this RNA-directed DNA endonuclease has been harnessed to target DNA sequences of interest. Here, we review the development of Cas9 as an important tool to not only edit the genomes of a number of different prokaryotic and eukaryotic species, but also as an efficient system for site-specific transcriptional repression or activation. Additionally, a specific Cas9 protein has been observed to target an RNA substrate, suggesting that Cas9 may have the ability to be programmed to target RNA as well. Cas proteins from other CRISPR/Cas subtypes may also be exploited in this regard. Thus, CRISPR/Cas systems represent an effective and versatile biotechnological tool, which will have significant impact on future advancements in genome engineering. © 2014 WILEY Periodicals, Inc.

  14. Programmable RNA recognition and cleavage by CRISPR/Cas9.

    PubMed

    O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

    2014-12-11

    The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

  15. Programmable RNA recognition and cleavage by CRISPR/Cas9

    PubMed Central

    O’Connell, Mitchell R.; Oakes, Benjamin L.; Sternberg, Samuel H.; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A.

    2014-01-01

    The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA:DNA complementarity to identify target sites for sequence-specific doublestranded DNA (dsDNA) cleavage1-5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, the protospacer adjacent motif (PAM), next to and on the strand opposite the 20-nucleotide target site in dsDNA4-7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in many cell types and organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalyzed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous mRNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable and tagless transcript recognition. PMID:25274302

  16. Crystal Structure of Streptococcus pyogenes Cas1 and Its Interaction with Csn2 in the Type II CRISPR-Cas System.

    PubMed

    Ka, Donghyun; Lee, Hasup; Jung, Yi-Deun; Kim, Kyunggon; Seok, Chaok; Suh, Nayoung; Bae, Euiyoung

    2016-01-05

    CRISPRs and Cas proteins constitute an RNA-guided microbial immune system against invading nucleic acids. Cas1 is a universal Cas protein found in all three types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition during CRISPR-mediated adaptive immunity. Here, we report the crystal structure of Streptococcus pyogenes Cas1 (SpCas1) in a type II CRISPR-Cas system and characterize its interaction with S. pyogenes Csn2 (SpCsn2). The SpCas1 structure reveals a unique conformational state distinct from type I Cas1 structures, resulting in a more extensive dimerization interface, a more globular overall structure, and a disruption of potential metal-binding sites for catalysis. We demonstrate that SpCas1 directly interacts with SpCsn2, and identify the binding interface and key residues for Cas complex formation. These results provide structural information for a type II Cas1 protein, and lay a foundation for studying multiprotein Cas complexes functioning in type II CRISPR-Cas systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Corrective Action Investigation Plan for Corrective Action Unit 168: Areas 25 and 26 Contaminated Materials and Waste Dumps, Nevada Test Site, Nevada (Rev. 0) includes Record of Technical Change No. 1 (dated 8/28/2002), Record of Technical Change No. 2 (dated 9/23/2002), and Record of Technical Change No. 3 (dated 6/2/2004)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    U.S. Department of Energy, National Nuclear Security Administration Nevada

    This Corrective Action Investigation Plan contains the U.S. Department of Energy, National Nuclear Security Administration Nevada Operations Office's approach to collect data necessary to evaluate corrective action alternatives appropriate for the closure of Corrective Action Unit 168 under the Federal Facility Agreement and Consent Order. Corrective Action Unit 168 consists of a group of twelve relatively diverse Corrective Action Sites (CASs 25-16-01, Construction Waste Pile; 25-16-03, MX Construction Landfill; 25-19-02, Waste Disposal Site; 25-23-02, Radioactive Storage RR Cars; 25-23-18, Radioactive Material Storage; 25-34-01, NRDS Contaminated Bunker; 25-34-02, NRDS Contaminated Bunker; CAS 25-23-13, ETL - Lab Radioactive Contamination; 25-99-16, USW G3;more » 26-08-01, Waste Dump/Burn Pit; 26-17-01, Pluto Waste Holding Area; 26-19-02, Contaminated Waste Dump No.2). These CASs vary in terms of the sources and nature of potential contamination. The CASs are located and/or associated wit h the following Nevada Test Site (NTS) facilities within three areas. The first eight CASs were in operation between 1958 to 1984 in Area 25 include the Engine Maintenance, Assembly, and Disassembly Facility; the Missile Experiment Salvage Yard; the Reactor Maintenance, Assembly, and Disassembly Facility; the Radioactive Materials Storage Facility; and the Treatment Test Facility Building at Test Cell A. Secondly, the three CASs located in Area 26 include the Project Pluto testing area that operated from 1961 to 1964. Lastly, the Underground Southern Nevada Well (USW) G3 (CAS 25-99-16), a groundwater monitoring well located west of the NTS on the ridgeline of Yucca Mountain, was in operation during the 1980s. Based on site history and existing characterization data obtained to support the data quality objectives process, contaminants of potential concern (COPCs) for CAU 168 are primarily radionuclide; however, the COPCs for several CASs were not defined. To address COPC

  18. Identification of genomic sites for CRISPR/Cas9-based genome editing in the Vitis vinifera genome

    USDA-ARS?s Scientific Manuscript database

    CRISPR/Cas9 has been recently demonstrated as an effective and popular genome editing tool for modifying genomes of human, animals, microorganisms, and plants. Success of such genome editing is highly dependent on the availability of suitable target sites in the genomes to be edited. Many specific t...

  19. Corrective Action Decision Document/Closure Report for Corrective Action Unit 371: Johnnie Boy Crater and Pin Stripe Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patrick Matthews

    undertaken that consist of implementing a use restriction and posting warning signs at each site. These use restrictions were recorded in the FFACO database; the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office (NNSA/NSO) Facility Information Management System; and the NNSA/NSO CAU/CAS files. Therefore, NNSA/NSO provides the following recommendations: • No further corrective actions are necessary for CAU 371. • A Notice of Completion to NNSA/NSO is requested from the Nevada Division of Environmental Protection for closure of CAU 371. • Corrective Action Unit 371 should be moved from Appendix III to Appendix IV of the FFACO.« less

  20. All-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated Multiplex Genome Engineering in Cultured Cells.

    PubMed

    Sakuma, Tetsushi; Sakamoto, Takuya; Yamamoto, Takashi

    2017-01-01

    CRISPR-Cas9 enables highly convenient multiplex genome engineering in cultured cells, because it utilizes generic Cas9 nuclease and an easily customizable single-guide RNA (sgRNA) for site-specific DNA double-strand break induction. We previously established a multiplex CRISPR-Cas9 assembly system for constructing an all-in-one vector simultaneously expressing multiple sgRNAs and Cas9 nuclease or other Cas9 variants including FokI-dCas9, which supersedes the wild-type Cas9 with regard to high specificity. In this chapter, we describe a streamlined protocol to design and construct multiplex CRISPR-Cas9 or FokI-dCas9 vectors, to introduce them into cultured cells by lipofection or electroporation, to enrich the genomically edited cells with a transient puromycin selection, to validate the mutation efficiency by Surveyor nuclease assay, and to perform off-target analyses. We show that our protocol enables highly efficient multiplex genome engineering even in hard-to-transfect HepG2 cells.

  1. Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

    PubMed Central

    Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

    2015-01-01

    Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

  2. Final corrective action study for the former CCC/USDA facility in Ramona, Kansas.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    LaFreniere, L. M.

    Past operations at a grain storage facility formerly leased and operated by the Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA) in Ramona, Kansas, resulted in low concentrations of carbon tetrachloride in groundwater that slightly exceed the regulatory standard in only one location. As requested by the Kansas Department of Health and Environment, the CCC/USDA has prepared a Corrective Action Study (CAS) for the facility. The CAS examines corrective actions to address groundwater impacted by the former CCC/USDA facility but not releases caused by other potential groundwater contamination sources in Ramona. Four remedial alternatives were considered in themore » CAS. The recommended remedial alternative in the CAS consists of Environmental Use Control to prevent the inadvertent use of groundwater as a water supply source, coupled with groundwater monitoring to verify the continued natural improvement in groundwater quality. The Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA) has directed Argonne National Laboratory to prepare a Corrective Action Study (CAS), consistent with guidance from the Kansas Department of Health and Environment (KDHE 2001a), for the CCC/USDA grain storage facility formerly located in Ramona, Kansas. This effort is pursuant to a KDHE (2007a) request. Although carbon tetrachloride levels at the Ramona site are low, they remain above the Kansas Tier 2 risk-based screening level (RBSL) and the U.S. Environmental Protection Agency (EPA) maximum contaminant level (MCL) of 5 {micro}g/L (Kansas 2003, 2004). In its request for the CAS, the KDHE (2007a) stated that, because of these levels, risk is associated with potential future exposure to contaminated groundwater. The KDHE therefore determined that additional measures are warranted to limit future use of the property and/or exposure to contaminated media as part of site closure. The KDHE further requested comparison of at least two

  3. Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.

    PubMed

    Subburaj, Saminathan; Chung, Sung Jin; Lee, Choongil; Ryu, Seuk-Min; Kim, Duk Hyoung; Kim, Jin-Soo; Bae, Sangsu; Lee, Geung-Joo

    2016-07-01

    Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

  4. Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems.

    PubMed

    Yamada, Mari; Watanabe, Yuto; Gootenberg, Jonathan S; Hirano, Hisato; Ran, F Ann; Nakane, Takanori; Ishitani, Ryuichiro; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

    2017-03-16

    The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Corrective Action Investigation Plan for Corrective Action Unit 410: Waste Disposal Trenches, Tonopah Test Range, Nevada, Revision 0 (includes ROTCs 1, 2, and 3)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NNSA /NV

    This Corrective Action Investigation Plan contains the U.S. Department of Energy, National Nuclear Security Administration Nevada Operations Office's approach to collect the data necessary to evaluate corrective action alternatives appropriate for the closure of Corrective Action Unit (CAU) 410 under the Federal Facility Agreement and Consent Order. Corrective Action Unit 410 is located on the Tonopah Test Range (TTR), which is included in the Nevada Test and Training Range (formerly the Nellis Air Force Range) approximately 140 miles northwest of Las Vegas, Nevada. This CAU is comprised of five Corrective Action Sites (CASs): TA-19-002-TAB2, Debris Mound; TA-21-003-TANL, Disposal Trench; TA-21-002-TAAL,more » Disposal Trench; 09-21-001-TA09, Disposal Trenches; 03-19-001, Waste Disposal Site. This CAU is being investigated because contaminants may be present in concentrations that could potentially pose a threat to human health and/or the environment, and waste may have been disposed of with out appropriate controls. Four out of five of these CASs are the result of weapons testing and disposal activities at the TTR, and they are grouped together for site closure based on the similarity of the sites (waste disposal sites and trenches). The fifth CAS, CAS 03-19-001, is a hydrocarbon spill related to activities in the area. This site is grouped with this CAU because of the location (TTR). Based on historical documentation and process know-ledge, vertical and lateral migration routes are possible for all CASs. Migration of contaminants may have occurred through transport by infiltration of precipitation through surface soil which serves as a driving force for downward migration of contaminants. Land-use scenarios limit future use of these CASs to industrial activities. The suspected contaminants of potential concern which have been identified are volatile organic compounds; semivolatile organic compounds; high explosives; radiological constituents including depleted

  6. CRISPR/Cas9 for genome editing: progress, implications and challenges.

    PubMed

    Zhang, Feng; Wen, Yan; Guo, Xiong

    2014-09-15

    Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system provides a robust and multiplexable genome editing tool, enabling researchers to precisely manipulate specific genomic elements, and facilitating the elucidation of target gene function in biology and diseases. CRISPR/Cas9 comprises of a nonspecific Cas9 nuclease and a set of programmable sequence-specific CRISPR RNA (crRNA), which can guide Cas9 to cleave DNA and generate double-strand breaks at target sites. Subsequent cellular DNA repair process leads to desired insertions, deletions or substitutions at target sites. The specificity of CRISPR/Cas9-mediated DNA cleavage requires target sequences matching crRNA and a protospacer adjacent motif locating at downstream of target sequences. Here, we review the molecular mechanism, applications and challenges of CRISPR/Cas9-mediated genome editing and clinical therapeutic potential of CRISPR/Cas9 in future. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Corrective Action Decision Document for Corrective Action Unit 562: Waste Systems Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mark Krause

    2010-08-01

    This Corrective Action Decision Document (CADD) presents information supporting the selection of corrective action alternatives (CAAs) leading to the closure of Corrective Action Unit (CAU) 562, Waste Systems, in Areas 2, 23, and 25 of the Nevada Test Site, Nevada. This complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; U.S. Department of Energy (DOE), Environmental Management; U.S. Department of Defense; and DOE, Legacy Management. Corrective Action Unit 562 comprises the following corrective action sites (CASs): • 02-26-11, Lead Shot • 02-44-02, Paint Spills and French Drainmore » • 02-59-01, Septic System • 02-60-01, Concrete Drain • 02-60-02, French Drain • 02-60-03, Steam Cleaning Drain • 02-60-04, French Drain • 02-60-05, French Drain • 02-60-06, French Drain • 02-60-07, French Drain • 23-60-01, Mud Trap Drain and Outfall • 23-99-06, Grease Trap • 25-60-04, Building 3123 Outfalls The purpose of this CADD is to identify and provide the rationale for the recommendation of CAAs for the 13 CASs within CAU 562. Corrective action investigation (CAI) activities were performed from July 27, 2009, through May 12, 2010, as set forth in the CAU 562 Corrective Action Investigation Plan. The purpose of the CAI was to fulfill the following data needs as defined during the data quality objective (DQO) process: • Determine whether COCs are present. • If COCs are present, determine their nature and extent. • Provide sufficient information and data to complete appropriate corrective actions. A data quality assessment (DQA) performed on the CAU 562 data demonstrated the quality and acceptability of the data for use in fulfilling the DQO data needs. Analytes detected during the CAI were evaluated against appropriate final action levels (FALs) to identify the COCs for each CAS. The results of the CAI identified COCs at 10 of the 13 CASs in CAU 562, and thus

  8. Addendum to the Corrective Action Decision Document/Closure Report for Corrective Action Unit 321: Area 22 Weather Station Fuel Storage Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the August 2001, Corrective Action Decision Document / Closure Report for Corrective Action Unit 321: Area 22 Weather Station Fuel Storage as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. The approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modificationmore » document, this addendum consists of: • This cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the cancellation of the UR for CAS 22-99-05, Fuel Storage Area. This UR was established as part of a Federal Facility Agreement and Consent Order (FFACO) corrective action and is based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since this UR was established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, this UR was re-evaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006c). This re-evaluation consisted of comparing the original data (used to define the need for the UR) to risk-based final action levels (FALs) developed using the current Industrial Sites RBCA process. The re-evaluation resulted in a recommendation to remove the UR

  9. [CRISPR/Cas system for genome editing in pluripotent stem cells].

    PubMed

    Vasil'eva, E A; Melino, D; Barlev, N A

    2015-01-01

    Genome editing systems based on site-specific nucleases became very popular for genome editing in modern bioengineering. Human pluripotent stem cells provide a unique platform for genes function study, disease modeling, and drugs testing. Consequently, technology for fast, accurate and well controlled genome manipulation is required. CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated) system could be employed for these purposes. This system is based on site-specific programmable nuclease Cas9. Numerous advantages of the CRISPR/Cas system and its successful application to human stem cells provide wide opportunities for genome therapy and regeneration medicine. In this publication, we describe and compare the main genome editing systems based on site-specific programmable nucleases and discuss opportunities and perspectives of the CRISPR/Cas system for application to pluripotent stem cells.

  10. Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

    PubMed

    Nuñez, James K; Harrington, Lucas B; Doudna, Jennifer A

    2016-03-18

    The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.

  11. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

    PubMed

    Sternberg, Samuel H; Redding, Sy; Jinek, Martin; Greene, Eric C; Doudna, Jennifer A

    2014-03-06

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

  12. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

    NASA Astrophysics Data System (ADS)

    Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

    2014-03-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

  13. Addendum to the Corrective Action Decision Document/Closure Report for Corrective Action Unit 529: Area 25 Contaminated Materials, Nevada Test Site, Nevada, Revision 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Krauss, Mark J

    This document constitutes an addendum to the Corrective Action Decision Document/Closure Report for Corrective Action Unit 529: Area 25 Contaminated Materials, Nevada Test Site, Nevada as described in the document Recommendations and Justifications To Remove Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Field Office Federal Facility Agreement and Consent Order dated September 2013. The Use Restriction (UR) Removal document was approved by the Nevada Division of Environmental Protection on October 16, 2013. The approval of the UR Removal document constituted approval of each of the recommended UR removals. In conformance with the URmore » Removal document, this addendum consists of: This page that refers the reader to the UR Removal document for additional information The cover, title, and signature pages of the UR Removal document The NDEP approval letter The corresponding section of the UR Removal document This addendum provides the documentation justifying the cancellation of the UR for CAS 25-23-17, Contaminated Wash (Parcel H). This UR was established as part of FFACO corrective actions and was based on the presence of total petroleum hydrocarbon diesel-range organics contamination at concentrations greater than the NDEP action level at the time of the initial investigation.« less

  14. The Impact on Student Achievement of When CAS Technology Is Introduced

    ERIC Educational Resources Information Center

    Driver, David

    2012-01-01

    When a Computer Algebra System (CAS) is used as a pedagogical and functional tool in class and as a functional tool in exams, its effect on student achievement can be quite profound. The timing of when students are first introduced to a CAS has an impact on gains in student achievement. In this action research project, the CAS calculator was…

  15. Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.

    PubMed

    Oakes, Benjamin L; Nadler, Dana C; Flamholz, Avi; Fellmann, Christof; Staahl, Brett T; Doudna, Jennifer A; Savage, David F

    2016-06-01

    The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzyme's binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the estrogen receptor-α ligand-binding domain. This protein showed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.

  16. Optimization of genome editing through CRISPR-Cas9 engineering.

    PubMed

    Zhang, Jian-Hua; Adikaram, Poorni; Pandey, Mritunjay; Genis, Allison; Simonds, William F

    2016-04-01

    CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats)-Cas9 (CRISPR associated protein 9) has rapidly become the most promising genome editing tool with great potential to revolutionize medicine. Through guidance of a 20 nucleotide RNA (gRNA), CRISPR-Cas9 finds and cuts target protospacer DNA precisely 3 base pairs upstream of a PAM (Protospacer Adjacent Motif). The broken DNA ends are repaired by either NHEJ (Non-Homologous End Joining) resulting in small indels, or by HDR (Homology Directed Repair) for precise gene or nucleotide replacement. Theoretically, CRISPR-Cas9 could be used to modify any genomic sequences, thereby providing a simple, easy, and cost effective means of genome wide gene editing. However, the off-target activity of CRISPR-Cas9 that cuts DNA sites with imperfect matches with gRNA have been of significant concern because clinical applications require 100% accuracy. Additionally, CRISPR-Cas9 has unpredictable efficiency among different DNA target sites and the PAM requirements greatly restrict its genome editing frequency. A large number of efforts have been made to address these impeding issues, but much more is needed to fully realize the medical potential of CRISPR-Cas9. In this article, we summarize the existing problems and current advances of the CRISPR-Cas9 technology and provide perspectives for the ultimate perfection of Cas9-mediated genome editing.

  17. Optimization of genome editing through CRISPR-Cas9 engineering

    PubMed Central

    Zhang, Jian-Hua; Adikaram, Poorni; Pandey, Mritunjay; Genis, Allison; Simonds, William F.

    2016-01-01

    ABSTRACT CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats)-Cas9 (CRISPR associated protein 9) has rapidly become the most promising genome editing tool with great potential to revolutionize medicine. Through guidance of a 20 nucleotide RNA (gRNA), CRISPR-Cas9 finds and cuts target protospacer DNA precisely 3 base pairs upstream of a PAM (Protospacer Adjacent Motif). The broken DNA ends are repaired by either NHEJ (Non-Homologous End Joining) resulting in small indels, or by HDR (Homology Directed Repair) for precise gene or nucleotide replacement. Theoretically, CRISPR-Cas9 could be used to modify any genomic sequences, thereby providing a simple, easy, and cost effective means of genome wide gene editing. However, the off-target activity of CRISPR-Cas9 that cuts DNA sites with imperfect matches with gRNA have been of significant concern because clinical applications require 100% accuracy. Additionally, CRISPR-Cas9 has unpredictable efficiency among different DNA target sites and the PAM requirements greatly restrict its genome editing frequency. A large number of efforts have been made to address these impeding issues, but much more is needed to fully realize the medical potential of CRISPR-Cas9. In this article, we summarize the existing problems and current advances of the CRISPR-Cas9 technology and provide perspectives for the ultimate perfection of Cas9-mediated genome editing. PMID:27340770

  18. Relationship between drug resistance and the clustered, regularly interspaced, short, palindromic repeat-associated protein genes cas1 and cas2 in Shigella from giant panda dung

    PubMed Central

    Ren, Lu; Deng, Lin-Hua; Zhang, Ri-Peng; Wang, Cheng-Dong; Li, De-Sheng; Xi, Li-Xin; Chen, Zhen-rong; Yang, Rui; Huang, Jie; Zeng, Yang-ru; Wu, Hong-Lin; Cao, San-Jie; Wu, Rui; Huang, Yong; Yan, Qi-Gui

    2017-01-01

    Abstract Background: To detect drug resistance in Shigella obtained from the dung of the giant panda, explore the factors leading to drug resistance in Shigella, understand the characteristics of clustered, regularly interspaced, short, palindromic repeats (CRISPR), and assess the relationship between CRISPR and drug resistance. Methods: We collected fresh feces from 27 healthy giant pandas in the Giant Panda Conservation base (Wolong, China). We identified the strains of Shigella in the samples by using nucleotide sequence analysis. Further, the Kirby-Bauer paper method was used to determine drug sensitivity of the Shigella strains. CRISPR-associated protein genes cas1 and cas2 in Shigella were detected by polymerase chain reaction (PCR), and the PCR products were sequenced and compared. Results: We isolated and identified 17 strains of Shigella from 27 samples, including 14 strains of Shigella flexneri, 2 strains of Shigella sonnei, and 1 strain of Shigella dysenteriae. Further, drug resistance to cefazolin, imipenem, and amoxicillin–clavulanic acid was identified as a serious problem, as multidrug-resistant strains were detected. Further, cas1 and cas2 showed different degrees of point mutations. Conclusion: The CRISPR system widely exists in Shigella and shares homology with that in Escherichia coli. The cas1 and cas 2 mutations contribute to the different levels of resistance. Point mutations at sites 3176455, 3176590, and 3176465 in cas1 (a); sites 3176989, 3176992, and 3176995 in cas1 (b); sites 3176156 and 3176236 in cas2 may affect the resistance of bacteria, cause emergence of multidrug resistance, and increase the types of drug resistance. PMID:28207509

  19. Relationship between drug resistance and the clustered, regularly interspaced, short, palindromic repeat-associated protein genes cas1 and cas2 in Shigella from giant panda dung.

    PubMed

    Ren, Lu; Deng, Lin-Hua; Zhang, Ri-Peng; Wang, Cheng-Dong; Li, De-Sheng; Xi, Li-Xin; Chen, Zhen-Rong; Yang, Rui; Huang, Jie; Zeng, Yang-Ru; Wu, Hong-Lin; Cao, San-Jie; Wu, Rui; Huang, Yong; Yan, Qi-Gui

    2017-02-01

    To detect drug resistance in Shigella obtained from the dung of the giant panda, explore the factors leading to drug resistance in Shigella, understand the characteristics of clustered, regularly interspaced, short, palindromic repeats (CRISPR), and assess the relationship between CRISPR and drug resistance. We collected fresh feces from 27 healthy giant pandas in the Giant Panda Conservation base (Wolong, China). We identified the strains of Shigella in the samples by using nucleotide sequence analysis. Further, the Kirby-Bauer paper method was used to determine drug sensitivity of the Shigella strains. CRISPR-associated protein genes cas1 and cas2 in Shigella were detected by polymerase chain reaction (PCR), and the PCR products were sequenced and compared. We isolated and identified 17 strains of Shigella from 27 samples, including 14 strains of Shigella flexneri, 2 strains of Shigella sonnei, and 1 strain of Shigella dysenteriae. Further, drug resistance to cefazolin, imipenem, and amoxicillin-clavulanic acid was identified as a serious problem, as multidrug-resistant strains were detected. Further, cas1 and cas2 showed different degrees of point mutations. The CRISPR system widely exists in Shigella and shares homology with that in Escherichia coli. The cas1 and cas 2 mutations contribute to the different levels of resistance. Point mutations at sites 3176455, 3176590, and 3176465 in cas1 (a); sites 3176989, 3176992, and 3176995 in cas1 (b); sites 3176156 and 3176236 in cas2 may affect the resistance of bacteria, cause emergence of multidrug resistance, and increase the types of drug resistance.

  20. Interacting Parallel Constructions of Knowledge in a CAS Context

    ERIC Educational Resources Information Center

    Kidron, Ivy; Dreyfus, Tommy

    2010-01-01

    We consider the influence of a CAS context on a learner's process of constructing a justification for the bifurcations in a logistic dynamical process. We describe how instrumentation led to cognitive constructions and how the roles of the learner and the CAS intertwine, especially close to the branching and combining of constructing actions. The…

  1. Breaking-Cas-interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes.

    PubMed

    Oliveros, Juan C; Franch, Mònica; Tabas-Madrid, Daniel; San-León, David; Montoliu, Lluis; Cubas, Pilar; Pazos, Florencio

    2016-07-08

    The CRISPR/Cas technology is enabling targeted genome editing in multiple organisms with unprecedented accuracy and specificity by using RNA-guided nucleases. A critical point when planning a CRISPR/Cas experiment is the design of the guide RNA (gRNA), which directs the nuclease and associated machinery to the desired genomic location. This gRNA has to fulfil the requirements of the nuclease and lack homology with other genome sites that could lead to off-target effects. Here we introduce the Breaking-Cas system for the design of gRNAs for CRISPR/Cas experiments, including those based in the Cas9 nuclease as well as others recently introduced. The server has unique features not available in other tools, including the possibility of using all eukaryotic genomes available in ENSEMBL (currently around 700), placing variable PAM sequences at 5' or 3' and setting the guide RNA length and the scores per nucleotides. It can be freely accessed at: http://bioinfogp.cnb.csic.es/tools/breakingcas, and the code is available upon request. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Corrective Action Investigation Plan for Corrective Action Unit 573: Alpha Contaminated Sites, Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    2014-05-01

    Corrective Action Unit (CAU) 573 is located in Area 5 of the Nevada National Security Site, which is approximately 65 miles northwest of Las Vegas, Nevada. CAU 573 is a grouping of sites where there has been a suspected release of contamination associated with non-nuclear experiments and nuclear testing. This document describes the planned investigation of CAU 573, which comprises the following corrective action sites (CASs): • 05-23-02, GMX Alpha Contaminated Area • 05-45-01, Atmospheric Test Site - Hamilton These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate andmore » recommend corrective action alternatives.« less

  3. Recruitment of CRISPR-Cas systems by Tn7-like transposons.

    PubMed

    Peters, Joseph E; Makarova, Kira S; Shmakov, Sergey; Koonin, Eugene V

    2017-08-29

    A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f , cas7f , and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.

  4. Addendum to the Streamlined Approach for Environmental Restoration Closure Report for Corrective Action Unit 454: Historical Undrground Storage Tank Release Sites, Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the April 1998, Streamlined Approach for Environmental Restoration Closure Report for Corrective Action Unit 454: Historical Underground Storage Tank Release Sites as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. The approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modificationmore » document, this addendum consists of: • This cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the cancellation of the UR for CAS 12-25-09, Spill 960722-02 (from UST 12-B-3). This UR was established as part of a Federal Facility Agreement and Consent Order (FFACO) corrective action and is based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since this UR was established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, this UR was re-evaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006c). This re-evaluation consisted of comparing the original data (used to define the need for the UR) to risk-based final action levels (FALs) developed using the current Industrial Sites RBCA process. The re-evaluation resulted in

  5. mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence.

    PubMed

    Finnigan, Gregory C; Thorner, Jeremy

    2016-07-07

    Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.e., to act at different genomic targets within the same nucleus. Currently, however, introducing concurrent changes at multiple loci involves: (i) identification of appropriate genomic sites, especially the availability of suitable PAM sequences; (ii) the design, construction, and expression of multiple sgRNA directed against those sites; (iii) potential difficulties in altering essential genes; and (iv) lingering concerns about "off-target" effects. We have devised a new approach that circumvents these drawbacks, as we demonstrate here using the yeast Saccharomyces cerevisiae First, any gene(s) of interest are flanked upstream and downstream with a single unique target sequence that does not normally exist in the genome. Thereafter, expression of one sgRNA and cotransformation with appropriate PCR fragments permits concomitant Cas9-mediated alteration of multiple genes (both essential and nonessential). The system we developed also allows for maintenance of the integrated, inducible Cas9-expression cassette or its simultaneous scarless excision. Our scheme-dubbed mCAL for " M: ultiplexing of C: as9 at A: rtificial L: oci"-can be applied to any organism in which the CRISPR/Cas9 methodology is currently being utilized. In principle, it can be applied to install synthetic sequences into the genome, to generate genomic libraries, and to program strains or cell lines so that they can be conveniently (and repeatedly

  6. Corrective Action Decision Document/Closure Report for Corrective Action Unit 105: Area 2 Yucca Flat Atmospheric Test Sites, Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    2013-09-01

    This Corrective Action Decision Document/Closure Report presents information supporting the closure of Corrective Action Unit (CAU) 105: Area 2 Yucca Flat Atmospheric Test Sites, Nevada National Security Site, Nevada. CAU 105 comprises the following five corrective action sites (CASs): -02-23-04 Atmospheric Test Site - Whitney Closure In Place -02-23-05 Atmospheric Test Site T-2A Closure In Place -02-23-06 Atmospheric Test Site T-2B Clean Closure -02-23-08 Atmospheric Test Site T-2 Closure In Place -02-23-09 Atmospheric Test Site - Turk Closure In Place The purpose of this Corrective Action Decision Document/Closure Report is to provide justification and documentation supporting the recommendation that nomore » further corrective action is needed for CAU 105 based on the implementation of the corrective actions. Corrective action investigation (CAI) activities were performed from October 22, 2012, through May 23, 2013, as set forth in the Corrective Action Investigation Plan for Corrective Action Unit 105: Area 2 Yucca Flat Atmospheric Test Sites; and in accordance with the Soils Activity Quality Assurance Plan, which establishes requirements, technical planning, and general quality practices.« less

  7. Corrective Action Decision Document/Corrective Action Plan for Corrective Action Unit 104: Area 7 Yucca Flat Atmospheric Test Sites Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patrick Matthews

    2012-10-01

    CAU 104 comprises the following corrective action sites (CASs): • 07-23-03, Atmospheric Test Site T-7C • 07-23-04, Atmospheric Test Site T7-1 • 07-23-05, Atmospheric Test Site • 07-23-06, Atmospheric Test Site T7-5a • 07-23-07, Atmospheric Test Site - Dog (T-S) • 07-23-08, Atmospheric Test Site - Baker (T-S) • 07-23-09, Atmospheric Test Site - Charlie (T-S) • 07-23-10, Atmospheric Test Site - Dixie • 07-23-11, Atmospheric Test Site - Dixie • 07-23-12, Atmospheric Test Site - Charlie (Bus) • 07-23-13, Atmospheric Test Site - Baker (Buster) • 07-23-14, Atmospheric Test Site - Ruth • 07-23-15, Atmospheric Test Site T7-4 •more » 07-23-16, Atmospheric Test Site B7-b • 07-23-17, Atmospheric Test Site - Climax These 15 CASs include releases from 30 atmospheric tests conducted in the approximately 1 square mile of CAU 104. Because releases associated with the CASs included in this CAU overlap and are not separate and distinguishable, these CASs are addressed jointly at the CAU level. The purpose of this CADD/CAP is to evaluate potential corrective action alternatives (CAAs), provide the rationale for the selection of recommended CAAs, and provide the plan for implementation of the recommended CAA for CAU 104. Corrective action investigation (CAI) activities were performed from October 4, 2011, through May 3, 2012, as set forth in the CAU 104 Corrective Action Investigation Plan.« less

  8. Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35.

    PubMed

    Zhao, Menglin; Wang, Jiaxian; Luo, Manyu; Luo, Han; Zhao, Meiqi; Han, Lei; Zhang, Mengxiao; Yang, Hui; Xie, Yueqing; Jiang, Hua; Feng, Lei; Lu, Huili; Zhu, Jianwei

    2018-07-01

    Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.

  9. RNA-dependent RNA targeting by CRISPR-Cas9

    PubMed Central

    Strutt, Steven C; Torrez, Rachel M; Kaya, Emine; Negrete, Oscar A

    2018-01-01

    Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications. PMID:29303478

  10. Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats.

    PubMed

    Shao, Yanjiao; Wang, Liren; Guo, Nana; Wang, Shengfei; Yang, Lei; Li, Yajing; Wang, Mingsong; Yin, Shuming; Han, Honghui; Zeng, Li; Zhang, Ludi; Hui, Lijian; Ding, Qiurong; Zhang, Jiqin; Geng, Hongquan; Liu, Mingyao; Li, Dali

    2018-05-04

    Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the Fah mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a Fah mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the Fah First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Corrective Action Decision Document/Closure Report for Corrective Action Unit 567: Miscellaneous Soil Sites - Nevada National Security Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    2014-12-01

    This Corrective Action Decision Document/Closure Report presents information supporting the closure of Corrective Action Unit (CAU) 567: Miscellaneous Soil Sites, Nevada National Security Site, Nevada. The purpose of this Corrective Action Decision Document/Closure Report is to provide justification and documentation supporting the recommendation that no further corrective action is needed for CAU 567 based on the implementation of the corrective actions. The corrective actions implemented at CAU 567 were developed based on an evaluation of analytical data from the CAI, the assumed presence of COCs at specific locations, and the detailed and comparative analysis of the CAAs. The CAAs weremore » selected on technical merit focusing on performance, reliability, feasibility, safety, and cost. The implemented corrective actions meet all requirements for the technical components evaluated. The CAAs meet all applicable federal and state regulations for closure of the site. Based on the implementation of these corrective actions, the DOE, National Nuclear Security Administration Nevada Field Office provides the following recommendations: • No further corrective actions are necessary for CAU 567. • The Nevada Division of Environmental Protection issue a Notice of Completion to the DOE, National Nuclear Security Administration Nevada Field Office for closure of CAU 567. • CAU 567 be moved from Appendix III to Appendix IV of the FFACO.« less

  12. Fusion of SpCas9 to E. coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells.

    PubMed

    Lin, Lin; Petersen, Trine Skov; Jensen, Kristopher Torp; Bolund, Lars; Kühn, Ralf; Luo, Yonglun

    2017-04-10

    Mammalian cells repair double-strand DNA breaks (DSB) by a range of different pathways following DSB induction by the engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9. While CRISPR-Cas9 thus enables predesigned modifications of the genome, applications of CRISPR-Cas9-mediated genome-editing are frequently hampered by the unpredictable and varying pathways for DSB repair in mammalian cells. Here we present a strategy of fusing Cas9 to recombinant proteins for fine-tuning of the DSB repair preferences in mammalian cells. By fusing Streptococcus Pyogenes Cas9 (SpCas9) to the recombinant protein A (Rec A, NP_417179.1) from Escherichia coli, we create a recombinant Cas9 protein (rSpCas9) which enhances the generation of indel mutations at DSB sites in mammalian cells, increases the frequency of DSB repair by homology-directed single-strand annealing (SSA), and represses homology-directed gene conversion by approximately 33%. Our study thus proves for the first time that fusing SpCas9 to recombinant proteins can influence the balance between DSB repair pathways in mammalian cells. This approach may form the basis for further investigations of the applications of recombinant Cas9 proteins to fine-tuning DSB repair pathways in eukaryotic cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13.

    PubMed

    Schindele, Patrick; Wolter, Felix; Puchta, Holger

    2018-04-30

    Currently, biology is revolutionized by ever growing applications of the CRISPR/Cas system. As discussed in this Review, new avenues have opened up for plant research and breeding by the use of the sequence-specific DNases Cas9 and Cas12 (formerly named Cpf1) and, more recently, the RNase Cas13 (formerly named C2c2). Although double strand break-induced gene editing based on error-prone nonhomologous end joining has been applied to obtain new traits, such as powdery mildew resistance in wheat or improved pathogen resistance and increased yield in tomato, improved technologies based on CRISPR/Cas for programmed change in plant genomes via homologous recombination have recently been developed. Cas9- and Cas12- mediated DNA binding is used to develop tools for many useful applications, such as transcriptional regulation or fluorescence-based imaging of specific chromosomal loci in plant genomes. Cas13 has recently been applied to degrade mRNAs and combat viral RNA replication. By the possibility to address multiple sequences with different guide RNAs and by the simultaneous use of different Cas proteins in a single cell, we should soon be able to achieve complex changes of plant metabolism in a controlled way. © 2018 Federation of European Biochemical Societies.

  14. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells

    PubMed Central

    Lee, Ciaran M; Cradick, Thomas J; Bao, Gang

    2016-01-01

    The clustered regularly-interspaced short palindromic repeats (CRISPR)—CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications. PMID:26782639

  15. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells.

    PubMed

    Lee, Ciaran M; Cradick, Thomas J; Bao, Gang

    2016-03-01

    The clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications.

  16. Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays.

    PubMed

    Lee, Hayun; Zhou, Yi; Taylor, David W; Sashital, Dipali G

    2018-04-05

    CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Cas9-Guide RNA Directed Genome Editing in Soybean[OPEN

    PubMed Central

    Li, Zhongsen; Liu, Zhan-Bin; Xing, Aiqiu; Moon, Bryan P.; Koellhoffer, Jessica P.; Huang, Lingxia; Ward, R. Timothy; Clifton, Elizabeth; Falco, S. Carl; Cigan, A. Mark

    2015-01-01

    Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites, DD20 and DD43 on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and one DD43 homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at the DD43 target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing. PMID:26294043

  18. Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

    PubMed Central

    Ethier, Sylvain; Schmeing, T. Martin; Dostie, Josée; Pelletier, Jerry

    2014-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5′NGG3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data. PMID:25275497

  19. CRISPR/Cas9 in Genome Editing and Beyond.

    PubMed

    Wang, Haifeng; La Russa, Marie; Qi, Lei S

    2016-06-02

    The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.

  20. Embryonal Fyn-associated substrate (EFS) and CASS4: The lesser-known CAS protein family members.

    PubMed

    Deneka, Alexander; Korobeynikov, Vladislav; Golemis, Erica A

    2015-10-01

    The CAS (Crk-associated substrate) adaptor protein family consists of four members: CASS1/BCAR1/p130Cas, CASS2/NEDD9/HEF1/Cas-L, CASS3/EFS/Sin and CASS4/HEPL. While CAS proteins lack enzymatic activity, they contain specific recognition and binding sites for assembly of larger signaling complexes that are essential for cell proliferation, survival, migration, and other processes. All family members are intermediates in integrin-dependent signaling pathways mediated at focal adhesions, and associate with FAK and SRC family kinases to activate downstream effectors regulating the actin cytoskeleton. Most studies of CAS proteins to date have been focused on the first two members, BCAR1 and NEDD9, with altered expression of these proteins now appreciated as influencing disease development and prognosis for cancer and other serious pathological conditions. For these family members, additional mechanisms of action have been defined in receptor tyrosine kinase (RTK) signaling, estrogen receptor signaling or cell cycle progression, involving discrete partner proteins such as SHC, NSP proteins, or AURKA. By contrast, EFS and CASS4 have been less studied, although structure-function analyses indicate they conserve many elements with the better-known family members. Intriguingly, a number of recent studies have implicated these proteins in immune system function, and the pathogenesis of developmental disorders, autoimmune disorders including Crohn's disease, Alzheimer's disease, cancer and other diseases. In this review, we summarize the current understanding of EFS and CASS4 protein function in the context of the larger CAS family group. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Closure report for Corrective Action Unit 211, Area 15 EPA Farm waste sites, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    1998-04-01

    This Closure Report summarizes the corrective actions which were completed at the Corrective Action Sites within Corrective Action Unit 211 Area 15 Farm Waste Sties at the Nevada Test Site. Current site descriptions, observations and identification of wastes removed are included on FFACO Corrective Action Site housekeeping closure verification forms.

  2. RNA-dependent RNA targeting by CRISPR-Cas9

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Strutt, Steven C.; Torrez, Rachel M.; Kaya, Emine

    Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo.more » We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. In conclusion, these results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.« less

  3. RNA-dependent RNA targeting by CRISPR-Cas9

    DOE PAGES

    Strutt, Steven C.; Torrez, Rachel M.; Kaya, Emine; ...

    2018-01-05

    Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo.more » We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. In conclusion, these results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.« less

  4. The host-encoded RNase E endonuclease as the crRNA maturation enzyme in a CRISPR-Cas subtype III-Bv system.

    PubMed

    Behler, Juliane; Sharma, Kundan; Reimann, Viktoria; Wilde, Annegret; Urlaub, Henning; Hess, Wolfgang R

    2018-03-01

    Specialized RNA endonucleases for the maturation of clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNAs (crRNAs) are critical in CRISPR-CRISPR-associated protein (Cas) defence mechanisms. The Cas6 and Cas5d enzymes are the RNA endonucleases in many class 1 CRISPR-Cas systems. In some class 2 systems, maturation and effector functions are combined within a single enzyme or maturation proceeds through the combined actions of RNase III and trans-activating CRISPR RNAs (tracrRNAs). Three separate CRISPR-Cas systems exist in the cyanobacterium Synechocystis sp. PCC 6803. Whereas Cas6-type enzymes act in two of these systems, the third, which is classified as subtype III-B variant (III-Bv), lacks cas6 homologues. Instead, the maturation of crRNAs proceeds through the activity of endoribonuclease E, leaving unusual 13- and 14-nucleotide-long 5'-handles. Overexpression of RNase E leads to overaccumulation and knock-down to the reduced accumulation of crRNAs in vivo, suggesting that RNase E is the limiting factor for CRISPR complex formation. Recognition by RNase E depends on a stem-loop in the CRISPR repeat, whereas base substitutions at the cleavage site trigger the appearance of secondary products, consistent with a two-step recognition and cleavage mechanism. These results suggest the adaptation of an otherwise very conserved housekeeping enzyme to accommodate new substrates and illuminate the impressive plasticity of CRISPR-Cas systems that enables them to function in particular genomic environments.

  5. Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii.

    PubMed

    Jiang, Wenzhi; Brueggeman, Andrew J; Horken, Kempton M; Plucinak, Thomas M; Weeks, Donald P

    2014-11-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10(9) cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  6. Using CRISPR-Cas9 to Study ERK Signaling in Drosophila.

    PubMed

    Forés, Marta; Papagianni, Aikaterini; Rodríguez-Muñoz, Laura; Jiménez, Gerardo

    2017-01-01

    Genome engineering using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated nuclease 9 (Cas9) technology is revolutionizing biomedical research. CRISPR-Cas9 enables precise editing of genes in a wide variety of cells and organisms, thereby accelerating molecular studies via targeted mutagenesis, epitope tagging, and other custom genetic modifications. Here, we illustrate the CRISPR-Cas9 methodology by focusing on Capicua (Cic), a nuclear transcriptional repressor directly phosphorylated and inactivated by ERK/MAPK. Specifically, we use CRISPR-Cas9 for targeting an ERK docking site of Drosophila Cic, thus generating ERK-insensitive mutants of this important signaling sensor.

  7. Corrective Action Decision Document for Corrective Action Unit 428: Area 3 Septic Waste Systems 1 and 5, Tonopah Test Range, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    U.S. Department of Energy, Nevada Operations Office

    2000-02-08

    This Corrective Action Decision Document identifies and rationalizes the US Department of Energy, Nevada Operations Office's selection of a recommended corrective action alternative (CAA) appropriate to facilitate the closure of Corrective Action Unit (CAU) 428, Septic Waste Systems 1 and 5, under the Federal Facility Agreement and Consent Order. Located in Area 3 at the Tonopah Test Range (TTR) in Nevada, CAU 428 is comprised of two Corrective Action Sites (CASs): (1) CAS 03-05-002-SW01, Septic Waste System 1 and (2) CAS 03-05-002- SW05, Septic Waste System 5. A corrective action investigation performed in 1999 detected analyte concentrations that exceeded preliminarymore » action levels; specifically, contaminants of concern (COCs) included benzo(a) pyrene in a septic tank integrity sample associated with Septic Tank 33-1A of Septic Waste System 1, and arsenic in a soil sample associated with Septic Waste System 5. During this investigation, three Corrective Action Objectives (CAOs) were identified to prevent or mitigate exposure to contents of the septic tanks and distribution box, to subsurface soil containing COCs, and the spread of COCs beyond the CAU. Based on these CAOs, a review of existing data, future use, and current operations in Area 3 of the TTR, three CAAs were developed for consideration: Alternative 1 - No Further Action; Alternative 2 - Closure in Place with Administrative Controls; and Alternative 3 - Clean Closure by Excavation and Disposal. These alternatives were evaluated based on four general corrective action standards and five remedy selection decision factors. Based on the results of the evaluation, the preferred CAA was Alternative 3. This alternative meets all applicable state and federal regulations for closure of the site and will eliminate potential future exposure pathways to the contaminated soils at the Area 3 Septic Waste Systems 1 and 5.« less

  8. DNA targeting specificity of RNA-guided Cas9 nucleases.

    PubMed

    Hsu, Patrick D; Scott, David A; Weinstein, Joshua A; Ran, F Ann; Konermann, Silvana; Agarwala, Vineeta; Li, Yinqing; Fine, Eli J; Wu, Xuebing; Shalem, Ophir; Cradick, Thomas J; Marraffini, Luciano A; Bao, Gang; Zhang, Feng

    2013-09-01

    The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

  9. CRISPR/Cas9-mediated correction of human genetic disease.

    PubMed

    Men, Ke; Duan, Xingmei; He, Zhiyao; Yang, Yang; Yao, Shaohua; Wei, Yuquan

    2017-05-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system (CRISPR/Cas9) provides a powerful tool for targeted genetic editing. Directed by programmable sequence-specific RNAs, this system introduces cleavage and double-stranded breaks at target sites precisely. Compared to previously developed targeted nucleases, the CRISPR/Cas9 system demonstrates several promising advantages, including simplicity, high specificity, and efficiency. Several broad genome-editing studies with the CRISPR/Cas9 system in different species in vivo and ex vivo have indicated its strong potential, raising hopes for therapeutic genome editing in clinical settings. Taking advantage of non-homologous end-joining (NHEJ) and homology directed repair (HDR)-mediated DNA repair, several studies have recently reported the use of CRISPR/Cas9 to successfully correct disease-causing alleles ranging from single base mutations to large insertions. In this review, we summarize and discuss recent preclinical studies involving the CRISPR/Cas9-mediated correction of human genetic diseases.

  10. Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.

    PubMed

    Bondy-Denomy, Joseph; Garcia, Bianca; Strum, Scott; Du, Mingjian; Rollins, MaryClare F; Hidalgo-Reyes, Yurima; Wiedenheft, Blake; Maxwell, Karen L; Davidson, Alan R

    2015-10-01

    The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR-Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR-Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor, providing the first example-to our knowledge-of modulation of CRISPR-Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR-Cas function.

  11. A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

    PubMed Central

    Chaikind, Brian; Bessen, Jeffrey L.; Thompson, David B.; Hu, Johnny H.; Liu, David R.

    2016-01-01

    We describe the development of ‘recCas9’, an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences. DNA sequencing reveals that recCas9 catalyzes guide RNA-dependent recombination in human cells with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases. PMID:27515511

  12. CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli

    PubMed Central

    Díez-Villaseñor, César; Guzmán, Noemí M.; Almendros, Cristóbal; García-Martínez, Jesús; Mojica, Francisco J.M.

    2013-01-01

    Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism. PMID:23445770

  13. CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli.

    PubMed

    Díez-Villaseñor, César; Guzmán, Noemí M; Almendros, Cristóbal; García-Martínez, Jesús; Mojica, Francisco J M

    2013-05-01

    Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism.

  14. The CRISPR/Cas revolution reaches the RNA world: Cas13, a new Swiss Army knife for plant biologists.

    PubMed

    Wolter, Felix; Puchta, Holger

    2018-06-01

    Application of the bacterial CRISPR/Cas systems to eukaryotes is revolutionizing biology. Cas9 and Cas12 (previously called Cpf1) are widely used as DNA nucleases for inducing site-specific DNA breaks for different kinds of genome engineering applications, and in their mutated forms as DNA-binding proteins to modify gene expression. Moreover, histone modifications, as well as cytosine methylation or base editing, were achieved with these systems in plants. Recently, with the discovery of the nuclease Cas13a (previously called C2c2), molecular biologists have obtained a system that enables sequence-specific cleavage of single-stranded RNA molecules. The latest experiments with this and also the alternative Cas13b system demonstrate that these proteins can be used in a similar manner in eukaryotes for RNA manipulation as Cas9 and Cas12 for DNA manipulations. The first application of Cas13a for post-transcriptional regulation of gene expression in plants has been reported. Recent results show that the system is also applicable for combating viral infection in plants. As single-stranded RNA viruses are by far the most abundant class of viruses in plants, the application of this system is of special promise for crops. More interesting applications are imminent for plant biologists, with nuclease dead versions of Cas13 enabling the ability to visualize RNA molecules in vivo, as well as to edit different kinds of RNA molecules at specific bases by deamination or to modify them by conjugation. Moreover, by combining DNA- and RNA-directed systems, the most complex of changes in plant metabolism might be achievable. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

  15. Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

    PubMed

    Heler, Robert; Samai, Poulami; Modell, Joshua W; Weiner, Catherine; Goldberg, Gregory W; Bikard, David; Marraffini, Luciano A

    2015-03-12

    Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

  16. Guidance on Initial Site Assessment at Corrective Action Sites

    EPA Pesticide Factsheets

    Guidance to be used to conduct Corrective Action site assessment efforts. Informs Resource Conservation and Recovery Act (RCRA) permit writers and enforcement officials of procedures to be used in conducting RCRA Facility Assessments.

  17. Rational design of a split-Cas9 enzyme complex.

    PubMed

    Wright, Addison V; Sternberg, Samuel H; Taylor, David W; Staahl, Brett T; Bardales, Jorge A; Kornfeld, Jack E; Doudna, Jennifer A

    2015-03-10

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

  18. Rational design of a split-Cas9 enzyme complex

    DOE PAGES

    Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; ...

    2015-02-23

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. The lobes do not interactmore » on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.« less

  19. Rational design of a split-Cas9 enzyme complex

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. The lobes do not interactmore » on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.« less

  20. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems

    PubMed Central

    Fonfara, Ines; Le Rhun, Anaïs; Chylinski, Krzysztof; Makarova, Kira S.; Lécrivain, Anne-Laure; Bzdrenga, Janek; Koonin, Eugene V.; Charpentier, Emmanuelle

    2014-01-01

    The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool. PMID:24270795

  1. Nuclear facility decommissioning and site remedial actions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Knox, N.P.; Webb, J.R.; Ferguson, S.D.

    1990-09-01

    The 394 abstracted references on environmental restoration, nuclear facility decommissioning, uranium mill tailings management, and site remedial actions constitute the eleventh in a series of reports prepared annually for the US Department of Energy's Remedial Action Programs. Citations to foreign and domestic literature of all types -- technical reports, progress reports, journal articles, symposia proceedings, theses, books, patents, legislation, and research project descriptions -- have been included. The bibliography contains scientific, technical, economic, regulatory, and legal information pertinent to the US Department of Energy's Remedial Action Programs. Major sections are (1) Surplus Facilities Management Program, (2) Nuclear Facilities Decommissioning, (3)more » Formerly Utilized Sites Remedial Action Programs, (4) Facilities Contaminated with Naturally Occurring Radionuclides, (5) Uranium Mill Tailings Remedial Action Program, (6) Grand Junction Remedial Action Program, (7) Uranium Mill Tailings Management, (8) Technical Measurements Center, (9) Remedial Action Program, and (10) Environmental Restoration Program. Within these categories, references are arranged alphabetically by first author. Those references having no individual author are listed by corporate affiliation or by publication title. Indexes are provided for author, corporate affiliation, title word, publication description, geographic location, subject category, and keywords. This report is a product of the Remedial Action Program Information Center (RAPIC), which selects and analyzes information on remedial actions and relevant radioactive waste management technologies.« less

  2. Editing Citrus Genome via SaCas9/sgRNA System

    PubMed Central

    Jia, Hongge; Xu, Jin; Orbović, Vladimir; Zhang, Yunzeng; Wang, Nian

    2017-01-01

    SaCas9/sgRNA, derived from Staphylococcus aureus, is an alternative system for genome editing to Streptococcus pyogenes SpCas9/sgRNA. The smaller SaCas9 recognizes a different protospacer adjacent motif (PAM) sequence from SpCas9. SaCas9/sgRNA has been employed to edit the genomes of Arabidopsis, tobacco and rice. In this study, we aimed to test its potential in genome editing of citrus. Transient expression of SaCas9/sgRNA in Duncan grapefruit via Xcc-facilitated agroinfiltration showed it can successfully modify CsPDS and Cs2g12470. Subsequently, binary vector GFP-p1380N-SaCas9/35S-sgRNA1:AtU6-sgRNA2 was developed to edit two target sites of Cs7g03360 in transgenic Carrizo citrange. Twelve GFP-positive Carrizo transformants were successfully established, designated as #Cz1 to #Cz12. Based on targeted next generation sequencing results, the mutation rates for the two targets ranged from 15.55 to 39.13% for sgRNA1 and 49.01 to 79.67% for sgRNA2. Therefore, SaCas9/sgRNA can be used as an alternative tool to SpCas9/sgRNA for citrus genome editing. PMID:29312390

  3. CRISPR-Cas9 technology and its application in haematological disorders

    PubMed Central

    Zhang, Han; McCarty, Nami

    2018-01-01

    Summary The recent advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system for precise genome editing has revolutionized methodologies in haematology and oncology studies. CRISPR-Cas9 technology can be used to remove and correct genes or mutations, and to introduce site-specific therapeutic genes in human cells. Inherited haematological disorders represent ideal targets for CRISPR-Cas9-mediated gene therapy. Correcting disease-causing mutations could alleviate disease-related symptoms in the near future. The CRISPR-Cas9 system is also a useful tool for delineating molecular mechanisms involving haematological malignancies. Prior to the use of CRISPR-Cas9-mediated gene correction in humans, appropriate delivery systems with higher efficiency and specificity must be identified, and ethical guidelines for applying the technology with controllable safety must be established. Here, the latest applications of CRISPR-Cas9 technology in haematological disorders, current challenges and future directions are reviewed and discussed. PMID:27619566

  4. CRISPR-Cas9 technology and its application in haematological disorders.

    PubMed

    Zhang, Han; McCarty, Nami

    2016-10-01

    The recent advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system for precise genome editing has revolutionized methodologies in haematology and oncology studies. CRISPR-Cas9 technology can be used to remove and correct genes or mutations, and to introduce site-specific therapeutic genes in human cells. Inherited haematological disorders represent ideal targets for CRISPR-Cas9-mediated gene therapy. Correcting disease-causing mutations could alleviate disease-related symptoms in the near future. The CRISPR-Cas9 system is also a useful tool for delineating molecular mechanisms involving haematological malignancies. Prior to the use of CRISPR-Cas9-mediated gene correction in humans, appropriate delivery systems with higher efficiency and specificity must be identified, and ethical guidelines for applying the technology with controllable safety must be established. Here, the latest applications of CRISPR-Cas9 technology in haematological disorders, current challenges and future directions are reviewed and discussed. © 2016 John Wiley & Sons Ltd.

  5. Addendum 2 to the Closure Report for Corrective Action Unit 358: Areas 18, 19, 20 Cellars/Mud Pits, Nevada Test Site, Nevada, Revison 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grant Evenson

    This document constitutes an addendum to the Closure Report for Corrective Action Unit 358: Areas 18, 19, 20 Cellars/Mud Pits, Nevada Test Site, Nevada, January 2004 as described in the document Supplemental Investigation Report for FFACO Use Restrictions, Nevada Test Site, Nevada (SIR) dated November 2008. The SIR document was approved by NDEP on December 5, 2008. The approval of the SIR document constituted approval of each of the recommended UR removals. In conformance with the SIR document, this addendum consists of: • This page that refers the reader to the SIR document for additional information • The cover, title,more » and signature pages of the SIR document • The NDEP approval letter • The corresponding section of the SIR document This addendum provides the documentation justifying the cancellation of the UR for CAS 19-09-05, Mud Pit. This UR was established as part of a Federal Facility Agreement and Consent Order (FFACO) corrective action and is based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996). Since this UR was established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, this UR was reevaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006). This re-evaluation consisted of comparing the original data (used to define the need for the UR) to risk-based final action levels (FALs) developed using the current Industrial Sites RBCA process. The re-evaluation resulted in a recommendation to remove the UR because contamination is not present at the site above the risk-based FALs. Requirements for inspecting and maintaining this UR will be canceled, and the postings and signage at this site will be removed. Fencing and posting may be present at this site that are unrelated to the FFACO UR such

  6. CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool

    PubMed Central

    del Sol Keyer, Maria; Wittbrodt, Joachim; Mateo, Juan L.

    2015-01-01

    Engineering of the CRISPR/Cas9 system has opened a plethora of new opportunities for site-directed mutagenesis and targeted genome modification. Fundamental to this is a stretch of twenty nucleotides at the 5’ end of a guide RNA that provides specificity to the bound Cas9 endonuclease. Since a sequence of twenty nucleotides can occur multiple times in a given genome and some mismatches seem to be accepted by the CRISPR/Cas9 complex, an efficient and reliable in silico selection and evaluation of the targeting site is key prerequisite for the experimental success. Here we present the CRISPR/Cas9 target online predictor (CCTop, http://crispr.cos.uni-heidelberg.de) to overcome limitations of already available tools. CCTop provides an intuitive user interface with reasonable default parameters that can easily be tuned by the user. From a given query sequence, CCTop identifies and ranks all candidate sgRNA target sites according to their off-target quality and displays full documentation. CCTop was experimentally validated for gene inactivation, non-homologous end-joining as well as homology directed repair. Thus, CCTop provides the bench biologist with a tool for the rapid and efficient identification of high quality target sites. PMID:25909470

  7. Addendum to the Closure Report for Corrective Action Unit 339: Area 12 Fleet Operations Steam Cleaning Discharge Area, Nevada Test Site, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grant Evenson

    This document constitutes an addendum to the Closure Report for CAU 339: Area 12 Fleet Operations Steam Cleaning Discharge Area Nevada Test Site, December 1997 as described in the document Supplemental Investigation Report for FFACO Use Restrictions, Nevada Test Site, Nevada (SIR) dated November 2008. The SIR document was approved by NDEP on December 5, 2008. The approval of the SIR document constituted approval of each of the recommended UR removals. In conformance with the SIR document, this addendum consists of: • This page that refers the reader to the SIR document for additional information • The cover, title, andmore » signature pages of the SIR document • The NDEP approval letter • The corresponding section of the SIR document This addendum provides the documentation justifying the cancellation of the UR for CAS 12-19-01, A12 Fleet Ops Steam Cleaning Efflu. This UR was established as part of a Federal Facility Agreement and Consent Order (FFACO) corrective action and is based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996). Since this UR was established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, this UR was reevaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006). This re-evaluation consisted of comparing the original data (used to define the need for the UR) to risk-based final action levels (FALs) developed using the current Industrial Sites RBCA process. The re-evaluation resulted in a recommendation to remove the UR because contamination is not present at the site above the risk-based FALs. Requirements for inspecting and maintaining this UR will be canceled, and the postings and signage at this site will be removed. Fencing and posting may be present at this site that are unrelated

  8. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector

    PubMed Central

    Kabadi, Ami M.; Ousterout, David G.; Hilton, Isaac B.; Gersbach, Charles A.

    2014-01-01

    Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types. PMID:25122746

  9. The CAS Classroom

    ERIC Educational Resources Information Center

    Garner, Sue

    2004-01-01

    The Victorian Curriculum and Assessment Authority (VCAA) Computer Algebra System (CAS)Pilot study (2001-2005) is monitoring the use of CAS in senior secondary mathematics. This article explores the author's experiences in the CAS classroom and delineates changes in teaching style, as a result of the introduction of CAS into the senior mathematics…

  10. Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System.

    PubMed

    Dong, Zhanqi; Dong, Feifan; Yu, Xinbo; Huang, Liang; Jiang, Yaming; Hu, Zhigang; Chen, Peng; Lu, Cheng; Pan, Minhui

    2018-01-01

    The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 ( ie-1 ) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

  11. Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System

    PubMed Central

    Dong, Zhanqi; Dong, Feifan; Yu, Xinbo; Huang, Liang; Jiang, Yaming; Hu, Zhigang; Chen, Peng; Lu, Cheng; Pan, Minhui

    2018-01-01

    The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 (ie-1) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment. PMID:29503634

  12. 76 FR 79545 - Cost Accounting Standards: Change to the CAS Applicability Threshold for the Inflation Adjustment...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-22

    ... Cost Accounting Standards: Change to the CAS Applicability Threshold for the Inflation Adjustment to... Federal Procurement Policy, Cost Accounting Standards Board. ACTION: Final rule. SUMMARY: The Office of Federal Procurement Policy (OFPP), Cost Accounting Standards (CAS) Board (Board), has adopted, without...

  13. Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease.

    PubMed

    Wolfs, Jason M; Hamilton, Thomas A; Lant, Jeremy T; Laforet, Marcon; Zhang, Jenny; Salemi, Louisa M; Gloor, Gregory B; Schild-Poulter, Caroline; Edgell, David R

    2016-12-27

    The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

  14. Addendum to the Closure Report for Corrective Action Unit 342: Area 23 Mercury Fire Training Pit Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the April 2000, Closure Report for Corrective Action Unit 342: Area 23 Mercury Fire Training Pit as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. The approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modification document, this addendum consists of:more » • This cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the cancellation of the UR for CAS 23-56-01, Former Mercury Fire Training Pit. This UR was established as part of a Federal Facility Agreement and Consent Order (FFACO) corrective action and is based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since this UR was established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, this UR was re-evaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006c). This re-evaluation consisted of comparing the original data (used to define the need for the UR) to risk-based final action levels (FALs) developed using the current Industrial Sites RBCA process. The re-evaluation resulted in a recommendation to remove the UR because contamination is not

  15. Corrective action investigation plan: Cactus Spring Waste Trenches. Revision 2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    This Correction Action Investigation Plan (CAIP) contains environmental sample collection objectives and logic for the CAU No. 426, which includes the Cactus Spring Waste Trenches, CAS No. RG-08-001-RG-CS. The Cactus Spring Waste Trenches are located at the Tonopah Test Range (TTR) which is part of the Nellis Air Force Range, approximately 255 kilometers (km) (140 miles [mi]) northwest of Las Vegas, Nevada, by air. The purpose of this investigation is to generate sufficient data to establish the types of waste buried in the trenches, identify the presence and nature of contamination, determine the vertical extent of contaminant migration below themore » Cactus Spring Waste Trenches, and determine the appropriate course of action for the site. The potential courses of action for the site are clean closure, closure in place (with or without remediation), or no further action.« less

  16. Corrective Action Investigation Plan for Corrective Action Unit 5: Landfills, Nevada Test Site, Nevada (Rev. No.: 0) includes Record of Technical Change No. 1 (dated 9/17/2002)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    IT Corporation, Las Vegas, NV

    This Corrective Action Investigation Plan contains the U.S. Department of Energy, National Nuclear Security Administration Nevada Operations Office's approach to collect the data necessary to evaluate corrective action alternatives appropriate for the closure of Corrective Action Unit (CAU) 5 under the Federal Facility Agreement and Consent Order. Corrective Action Unit 5 consists of eight Corrective Action Sites (CASs): 05-15-01, Sanitary Landfill; 05-16-01, Landfill; 06-08-01, Landfill; 06-15-02, Sanitary Landfill; 06-15-03, Sanitary Landfill; 12-15-01, Sanitary Landfill; 20-15-01, Landfill; 23-15-03, Disposal Site. Located between Areas 5, 6, 12, 20, and 23 of the Nevada Test Site (NTS), CAU 5 consists of unlined landfillsmore » used in support of disposal operations between 1952 and 1992. Large volumes of solid waste were produced from the projects which used the CAU 5 landfills. Waste disposed in these landfills may be present without appropriate controls (i.e., use restrictions, adequate cover) and hazardous and/or radioactive constituents may be present at concentrations and locations that could potentially pose a threat to human health and/or the environment. During the 1992 to 1995 time frame, the NTS was used for various research and development projects including nuclear weapons testing. Instead of managing solid waste at one or two disposal sites, the practice on the NTS was to dispose of solid waste in the vicinity of the project. A review of historical documentation, process knowledge, personal interviews, and inferred activities associated with this CAU identified the following as potential contaminants of concern: volatile organic compounds, semivolatile organic compounds, polychlorinated biphenyls, pesticides, petroleum hydrocarbons (diesel- and gasoline-range organics), Resource Conservation and Recovery Act Metals, plus nickel and zinc. A two-phase approach has been selected to collect information and generate data to satisfy needed resolution

  17. Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition

    PubMed Central

    Rollie, Clare; Schneider, Stefanie; Brinkmann, Anna Sophie; Bolt, Edward L; White, Malcolm F

    2015-01-01

    The adaptive prokaryotic immune system CRISPR-Cas provides RNA-mediated protection from invading genetic elements. The fundamental basis of the system is the ability to capture small pieces of foreign DNA for incorporation into the genome at the CRISPR locus, a process known as Adaptation, which is dependent on the Cas1 and Cas2 proteins. We demonstrate that Cas1 catalyses an efficient trans-esterification reaction on branched DNA substrates, which represents the reverse- or disintegration reaction. Cas1 from both Escherichia coli and Sulfolobus solfataricus display sequence specific activity, with a clear preference for the nucleotides flanking the integration site at the leader-repeat 1 boundary of the CRISPR locus. Cas2 is not required for this activity and does not influence the specificity. This suggests that the inherent sequence specificity of Cas1 is a major determinant of the adaptation process. DOI: http://dx.doi.org/10.7554/eLife.08716.001 PMID:26284603

  18. The CRISPR-Cas system - from bacterial immunity to genome engineering.

    PubMed

    Czarnek, Maria; Bereta, Joanna

    2016-09-01

    Precise and efficient genome modifications present a great value in attempts to comprehend the roles of particular genes and other genetic elements in biological processes as well as in various pathologies. In recent years novel methods of genome modification known as genome editing, which utilize so called "programmable" nucleases, came into use. A true revolution in genome editing has been brought about by the introduction of the CRISP-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) system, in which one of such nucleases, i.e. Cas9, plays a major role. This system is based on the elements of the bacterial and archaeal mechanism responsible for acquired immunity against phage infections and transfer of foreign genetic material. Microorganisms incorporate fragments of foreign DNA into CRISPR loci present in their genomes, which enables fast recognition and elimination of future infections. There are several types of CRISPR-Cas systems among prokaryotes but only elements of CRISPR type II are employed in genome engineering. CRISPR-Cas type II utilizes small RNA molecules (crRNA and tracrRNA) to precisely direct the effector nuclease - Cas9 - to a specific site in the genome, i.e. to the sequence complementary to crRNA. Cas9 may be used to: (i) introduce stable changes into genomes e.g. in the process of generation of knock-out and knock-in animals and cell lines, (ii) activate or silence the expression of a gene of interest, and (iii) visualize specific sites in genomes of living cells. The CRISPR-Cas-based tools have been successfully employed for generation of animal and cell models of a number of diseases, e.g. specific types of cancer. In the future, the genome editing by programmable nucleases may find wide application in medicine e.g. in the therapies of certain diseases of genetic origin and in the therapy of HIV-infected patients.

  19. Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage

    PubMed Central

    Josephs, Eric A.; Kocak, D. Dewran; Fitzgibbon, Christopher J.; McMenemy, Joshua; Gersbach, Charles A.; Marszalek, Piotr E.

    2015-01-01

    CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9's ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9's off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex. PMID:26384421

  20. Addendum to the Closure Report for Corrective Action Unit 357: Mud Pits and Waste Dump, Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Krauss, Mark J

    This document constitutes an addendum to the Closure Report for Corrective Action Unit 357: Mud Pits and Waste Dump, Nevada Test Site, Nevada as described in the document Recommendations and Justifications To Remove Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Field Office Federal Facility Agreement and Consent Order dated September 2013. The Use Restriction Removal document was approved by the Nevada Division of Environmental Protection on October 16, 2013. The approval of the UR Removal document constituted approval of each of the recommended UR removals. In conformance with the UR Removal document, thismore » addendum consists of: This page that refers the reader to the UR Removal document for additional information The cover, title, and signature pages of the UR Removal document The NDEP approval letter The corresponding section of the UR Removal document This addendum provides the documentation justifying the cancellation of the UR for CAS 04-26-03, Lead Bricks. This UR was established as part of FFACO corrective actions and was based on the presence of lead contamination at concentrations greater than the action level established at the time of the initial investigation.« less

  1. Corrective action investigation plan for Corrective Action Unit Number 423: Building 03-60 Underground Discharge Point, Tonopah Test Range, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    1997-10-27

    This Corrective Action Investigation Plan (CAIP) contains the environmental sample collection objectives and the criteria for conducting site investigation activities at Corrective Action Unit (CAU) Number 423, the Building 03-60 Underground Discharge Point (UDP), which is located in Area 3 at the Tonopah Test Range (TTR). The TTR, part of the Nellis Air Force Range, is approximately 225 kilometers (140 miles) northwest of Las Vegas, Nevada. CAU Number 423 is comprised of only one Corrective Action Site (CAS) which includes the Building 03-60 UDP and an associated discharge line extending from Building 03-60 to a point approximately 73 meters (240more » feet) northwest. The UDP was used between approximately 1965 and 1990 to dispose of waste fluids from the Building 03-60 automotive maintenance shop. It is likely that soils surrounding the UDP have been impacted by oil, grease, cleaning supplies and solvents as well as waste motor oil and other automotive fluids released from the UDP.« less

  2. The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET.

    PubMed

    Yang, Mengyi; Peng, Sijia; Sun, Ruirui; Lin, Jingdi; Wang, Nan; Chen, Chunlai

    2018-01-09

    Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  3. CRISPR/Cas9 Inhibits Multiple Steps of HIV-1 Infection.

    PubMed

    Yin, Lijuan; Hu, Siqi; Mei, Shan; Sun, Hong; Xu, Fengwen; Li, Jian; Zhu, Weijun; Liu, Xiaoman; Zhao, Fei; Zhang, Di; Cen, Shan; Liang, Chen; Guo, Fei

    2018-05-09

    CRISPR/Cas9 is an adaptive immune system where bacteria and archaea have evolved to resist the invading viruses and plasmid DNA by creating site-specific double-strand breaks in DNA. This study tested this gene editing system in inhibiting human immunodeficiency virus type 1 (HIV-1) infection by targeting the viral long terminal repeat and the gene coding sequences. Strong inhibition of HIV-1 infection by Cas9/gRNA was observed, which resulted not only from insertions and deletions (indels) that were introduced into viral DNA due to Cas9 cleavage, but also from the marked decrease in the levels of the late viral DNA products and the integrated viral DNA. This latter defect might have reflected the degradation of viral DNA that has not been immediately repaired after Cas9 cleavage. It was further observed that Cas9, when solely located in the cytoplasm, inhibits HIV-1 as strongly as the nuclear Cas9, except that the cytoplasmic Cas9 does not act on the integrated HIV-1 DNA and thus cannot be used to excise the latent provirus. Together, the results suggest that Cas9/gRNA is able to target and edit HIV-1 DNA both in the cytoplasm and in the nucleus. The inhibitory effect of Cas9 on HIV-1 is attributed to both the indels in viral DNA and the reduction in the levels of viral DNA.

  4. RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?

    PubMed

    Gasiunas, Giedrius; Siksnys, Virginijus

    2013-11-01

    Tailor-made nucleases for precise genome modification, such as zinc finger or TALE nucleases, currently represent the state-of-the-art for genome editing. These nucleases combine a programmable protein module which guides the enzyme to the target site with a nuclease domain which cuts DNA at the addressed site. Reprogramming of these nucleases to cut genomes at specific locations requires major protein engineering efforts. RNA-guided DNA endonuclease Cas9 of the type II (clustered regularly interspaced short palindromic repeat) CRISPR-Cas system uses CRISPR RNA (crRNA) as a guide to locate the DNA target and the Cas9 protein to cut DNA. Easy programmability of the Cas9 endonuclease using customizable RNAs brings unprecedented flexibility and versatility for targeted genome modification. We highlight the potential of the Cas9 RNA-guided DNA endonuclease as a novel tool for genome surgery, and discuss possible constraints and future prospects. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. CRISPR/Cas-mediated knock-in via non-homologous end-joining in the crustacean Daphnia magna

    PubMed Central

    Kumagai, Hitoshi; Nakanishi, Takashi; Matsuura, Tomoaki; Kato, Yasuhiko

    2017-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is widely used for mediating the knock-in of foreign DNA into the genomes of various organisms. Here, we report a process of CRISPR/Cas-mediated knock-in via non-homologous end joining by the direct injection of Cas9/gRNA ribonucleoproteins (RNPs) in the crustacean Daphnia magna, which is a model organism for studies on toxicology, ecology, and evolution. First, we confirmed the cleavage activity of Cas9 RNPs comprising purified Cas9 proteins and gRNAs in D. magna. We used a gRNA that targets exon 10 of the eyeless gene. Cas9 proteins were incubated with the gRNAs and the resulting Cas9 RNPs were injected into D. magna eggs, which led to a typical phenotype of the eyeless mutant, i.e., eye deformity. The somatic and heritable mutagenesis efficiencies were up to 96% and 40%, respectively. Second, we tested the CRISPR/Cas-mediated knock-in of a plasmid by the injection of Cas9 RNPs. The donor DNA plasmid harboring the fluorescent reporter gene was designed to contain the gRNA recognition site. The co-injection of Cas9 RNPs together with the donor DNAs resulted in generation of one founder animal that produced fluorescent progenies. This transgenic Daphnia had donor DNA at the targeted genomic site, which suggested the concurrent cleavage of the injected plasmid DNA and genomic DNA. Owing to its simplicity and ease of experimental design, we suggest that the CRISPR/Cas-mediated knock-in method represents a promising tool for studying functional genomics in D. magna. PMID:29045453

  6. CRISPR/Cas-mediated knock-in via non-homologous end-joining in the crustacean Daphnia magna.

    PubMed

    Kumagai, Hitoshi; Nakanishi, Takashi; Matsuura, Tomoaki; Kato, Yasuhiko; Watanabe, Hajime

    2017-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is widely used for mediating the knock-in of foreign DNA into the genomes of various organisms. Here, we report a process of CRISPR/Cas-mediated knock-in via non-homologous end joining by the direct injection of Cas9/gRNA ribonucleoproteins (RNPs) in the crustacean Daphnia magna, which is a model organism for studies on toxicology, ecology, and evolution. First, we confirmed the cleavage activity of Cas9 RNPs comprising purified Cas9 proteins and gRNAs in D. magna. We used a gRNA that targets exon 10 of the eyeless gene. Cas9 proteins were incubated with the gRNAs and the resulting Cas9 RNPs were injected into D. magna eggs, which led to a typical phenotype of the eyeless mutant, i.e., eye deformity. The somatic and heritable mutagenesis efficiencies were up to 96% and 40%, respectively. Second, we tested the CRISPR/Cas-mediated knock-in of a plasmid by the injection of Cas9 RNPs. The donor DNA plasmid harboring the fluorescent reporter gene was designed to contain the gRNA recognition site. The co-injection of Cas9 RNPs together with the donor DNAs resulted in generation of one founder animal that produced fluorescent progenies. This transgenic Daphnia had donor DNA at the targeted genomic site, which suggested the concurrent cleavage of the injected plasmid DNA and genomic DNA. Owing to its simplicity and ease of experimental design, we suggest that the CRISPR/Cas-mediated knock-in method represents a promising tool for studying functional genomics in D. magna.

  7. Therapeutic applications of CRISPR/Cas9 system in gene therapy.

    PubMed

    Mollanoori, Hasan; Teimourian, Shahram

    2018-06-01

    Gene therapy is based on the principle of the genetic manipulation of DNA or RNA for treating and preventing human diseases. The clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease9 (CRISPR/Cas9) system, derived from the acquired immune system in bacteria and archaea, has provided a new tool for accurate manipulation of genomic sequence to attain a therapeutic result. The advantage of CRISPR which made it an easy and flexible tool for diverse genome editing purposes is that a single protein (Cas9) complex with 2 short RNA sequences, function as a site-specific endonuclease. Recently, application of CRISPR/Cas9 system has become popular for therapeutic aims such as gene therapy. In this article, we review the fundamental mechanisms of CRISPR-Cas9 function and summarize preclinical CRISPR-mediated gene therapy reports on a wide variety of disorders.

  8. Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity

    PubMed Central

    Tycko, Josh; Myer, Vic E.; Hsu, Patrick D.

    2016-01-01

    Summary Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance. PMID:27494557

  9. Site Rehabilitation Completion Report with No Further Action Proposal for the Northeast Site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Daniel, Joe; Tabor, Charles; Survochak, Scott

    2013-05-01

    The purpose of this Site Rehabilitation Completion Report is to present the post-active-remediation monitoring results for the Northeast Site and to propose No Further Action with Controls. This document includes information required by Chapter 62-780.750(4)(d), 62-780.750(6), and 62-780.600(8)(a)27 Florida Administrative Code (F.A.C.). The Closure Monitoring Plan for the Northeast Site and 4.5 Acre Site (DOE 2009a) describes the approach for post-active-remediation monitoring. The Young - Rainey Science, Technology, and Research Center (STAR Center) is a former U.S. Department of Energy (DOE) facility constructed in the mid-1950s. The 99-acre STAR Center is located in Largo, Florida. The Northeast Site is locatedmore » in the northeast corner of the STAR Center. The Northeast Site meets all the requirements for an RMO II closure—No Further Action with Controls. DOE is nearing completion of a restrictive covenant for the Northeast Site. DOE has completed post-active-remediation monitoring at the Northeast Site as of September 2012. No additional monitoring will be conducted.« less

  10. Molecular basis, applications and challenges of CRISPR/Cas9: a continuously evolving tool for genome editing.

    PubMed

    D'Agostino, Ylenia; D'Aniello, Salvatore

    2017-07-01

    The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system is a recently discovered tool for genome editing that has quickly revolutionized the ability to generate site-specific mutations in a wide range of animal models, including nonhuman primates. Indeed, a significant number of scientific reports describing single or multiplex guide RNA microinjection, double-nicking strategies, site-specific knock-in and conditional knock-out have been published in less than three years. However, despite the great potential of this new technology, there are some limitations because of the presence of off-target genomic sites, which must be taken into consideration. To address this issue, various research teams have tried to improve the efficiency of the system through enzymatic modifications of the Cas9 protein or by the introduction of alternative strategies. Although several review articles are available that singly describe the molecular mechanism(s), applications and challenges of each of these strategies, a concise compilation of approaches is lacking. In the current review, we describe and evaluate most CRISPR/Cas9 approaches available at present, describing both mechanism of action, in addition to advantages or disadvantages. The primary goal of this work is to serve as a guide for not skilled researchers, facilitating the selection of the best strategy to target their gene of interest and allowing optimization of particular applications to the specific aims of the study. The present article also offers a unique perspective, focusing on the fact that CRISPR technology is opening a new genomic era, providing the means to manipulate specific genes in a targeted manner in all animal models, an endeavor previously considered to be difficult. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. Remedial Action Plan and site design for stabilization of the inactive uranium mill tailings site at Durango, Colorado: Remedial action selection report

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Not Available

    1991-12-01

    The uranium mill tailings site near Durango, Colorado, was one of 24 inactive uranium mill sites designated to be remediated by the US Department of Energy (DOE) under the Uranium Mill Tailings Radiation Control Act of 1978 (UMTRCA). Part of the UMTRCA requires that the US Nuclear Regulatory Commission (NRC) concur with the DOE's Remedial Action Plan (RAP) and certify that the remedial action conducted at the site complies with the standards promulgated by the US Environmental Protection Agency (EPA). Included in the RAP is this Remedial Action Selection Report (RAS), which has been developed to serve a two-fold purpose.more » First, it describes the activities that have been conducted by the DOE to accomplish remediation and long-term stabilization and control of the radioactive materials at the inactive uranium mill processing site near Durango, Colorado. Secondly, this document and the rest of the RAP, upon concurrence and execution by the DOE, the State of Colorado, and the NRC, become Appendix B of the Cooperative Agreement between the DOE and the State of Colorado.« less

  12. Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b.

    PubMed

    Richter, Hagen; Rompf, Judith; Wiegel, Julia; Rau, Kristina; Randau, Lennart

    2017-11-01

    CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection. Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays. The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate. Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Corrective Action Investigation Plan for Corrective Action Unit 569: Area 3 Yucca Flat Atmospheric Test Sites Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patrick Matthews; Christy Sloop

    2012-02-01

    Corrective Action Unit (CAU) 569 is located in Area 3 of the Nevada National Security Site, which is approximately 65 miles northwest of Las Vegas, Nevada. Corrective Action Unit 569 comprises the nine numbered corrective action sites (CASs) and one newly identified site listed below: (1) 03-23-09, T-3 Contamination Area (hereafter referred to as Annie, Franklin, George, and Moth); (2) 03-23-10, T-3A Contamination Area (hereafter referred to as Harry and Hornet); (3) 03-23-11, T-3B Contamination Area (hereafter referred to as Fizeau); (4) 03-23-12, T-3S Contamination Area (hereafter referred to as Rio Arriba); (5) 03-23-13, T-3T Contamination Area (hereafter referred tomore » as Catron); (6) 03-23-14, T-3V Contamination Area (hereafter referred to as Humboldt); (7) 03-23-15, S-3G Contamination Area (hereafter referred to as Coulomb-B); (8) 03-23-16, S-3H Contamination Area (hereafter referred to as Coulomb-A); (9) 03-23-21, Pike Contamination Area (hereafter referred to as Pike); and (10) Waste Consolidation Site 3A. Because CAU 569 is a complicated site containing many types of releases, it was agreed during the data quality objectives (DQO) process that these sites will be grouped. These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives (CAAs). Additional information will be obtained by conducting a corrective action investigation before evaluating CAAs and selecting the appropriate corrective action for each study group. The results of the field investigation will support a defensible evaluation of viable CAAs that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the DQOs developed on September 26, 2011, by representatives of the Nevada Division of Environmental Protection and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office. The

  14. CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes.

    PubMed

    Koonin, Eugene V; Makarova, Kira S

    2013-05-01

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated genes) is an adaptive immunity system in bacteria and archaea that functions via a distinct self-non-self recognition mechanism that is partially analogous to the mechanism of eukaryotic RNA interference (RNAi). The CRISPR-Cas system incorporates fragments of virus or plasmid DNA into the CRISPR repeat cassettes and employs the processed transcripts of these spacers as guide RNAs to cleave the cognate foreign DNA or RNA. The Cas proteins, however, are not homologous to the proteins involved in RNAi and comprise numerous, highly diverged families. The majority of the Cas proteins contain diverse variants of the RNA recognition motif (RRM), a widespread RNA-binding domain. Despite the fast evolution that is typical of the cas genes, the presence of diverse versions of the RRM in most Cas proteins provides for a simple scenario for the evolution of the three distinct types of CRISPR-cas systems. In addition to several proteins that are directly implicated in the immune response, the cas genes encode a variety of proteins that are homologous to prokaryotic toxins that typically possess nuclease activity. The predicted toxins associated with CRISPR-Cas systems include the essential Cas2 protein, proteins of COG1517 that, in addition to a ligand-binding domain and a helix-turn-helix domain, typically contain different nuclease domains and several other predicted nucleases. The tight association of the CRISPR-Cas immunity systems with predicted toxins that, upon activation, would induce dormancy or cell death suggests that adaptive immunity and dormancy/suicide response are functionally coupled. Such coupling could manifest in the persistence state being induced and potentially providing conditions for more effective action of the immune system or in cell death being triggered when immunity fails.

  15. Spacer capture and integration by a type I-F Cas1-Cas2-3 CRISPR adaptation complex.

    PubMed

    Fagerlund, Robert D; Wilkinson, Max E; Klykov, Oleg; Barendregt, Arjan; Pearce, F Grant; Kieper, Sebastian N; Maxwell, Howard W R; Capolupo, Angela; Heck, Albert J R; Krause, Kurt L; Bostina, Mihnea; Scheltema, Richard A; Staals, Raymond H J; Fineran, Peter C

    2017-06-27

    CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1-Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas1 4 -Cas2-3 2 complex from Pectobacterium atrosepticum with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1-Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1-Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3'-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1-Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1-Cas2-3 adaptation complexes and their integration mechanism.

  16. CORRECTIVE ACTION PLAN FOR CORRECTIVE ACTION UNIT 516: SEPTIC SYSTEMS AND DISCHARGE POINTS, NEVADA TEST SITE, NEVADA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    BECHTEL NEVADA; U.S. DEPARTMENT OF ENERGY, NATIONAL NUCLEAR SECURITY ADMINISTRATION NEVADA SITE OFFICE

    2005-08-01

    Corrective Action Unit (CAU) 516, Septic Systems and Discharge Points, is listed in the ''Federal Facility Agreement and Consent Order'' (FFACO) of 1996 (FFACO, 1996). CAU 516 consists of six Corrective Action Sites (CASs) located in Areas 3, 6, and 22 of the Nevada Test Site (NTS), which is located approximately 65 miles northwest of Las Vegas, Nevada (Figure 1). CAU 516 is comprised of the following six CASs: (1) 03-59-01 Building 3C-36 Septic System; (2) 03-59-02 Building 3C-45 Septic System; (3) 06-51-01 Sump and Piping; (4) 06-51-02 Clay Pipe and Debris; (5) 06-51-03 Clean-Out Box and Piping; and (6)more » 22-19-04 Vehicle Decontamination Area. Details on site history and site characterization results for CAU 516 are provided in the approved Corrective Action Investigation Plan (CAIP), (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2003), and the approved Corrective Action Decision Document (CADD) (NNSA/NSO, 2004).« less

  17. A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation

    PubMed Central

    Farasat, Iman; Salis, Howard M.

    2016-01-01

    The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical and engineering applications. The CRISPR/Cas9 (Type II) system binds and cuts DNA using guide RNAs, though the variables that control its on-target and off-target activity remain poorly characterized. Here, we develop and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, Cas9 and crRNA expression levels, organisms and growth conditions, and experimental conditions collectively control the dynamics of dCas9-based binding and Cas9-based cleavage at all DNA sites with both canonical and non-canonical PAMs. We combine statistical thermodynamics and kinetics to model Cas9:crRNA complex formation, diffusion, site selection, reversible R-loop formation, and cleavage, using large amounts of structural, biochemical, expression, and next-generation sequencing data to determine kinetic parameters and develop free energy models. Our results identify DNA supercoiling as a novel mechanism controlling Cas9 binding. Using the model, we predict Cas9 off-target binding frequencies across the lambdaphage and human genomes, and explain why Cas9’s off-target activity can be so high. With this improved understanding, we propose several rules for designing experiments for minimizing off-target activity. We also discuss the implications for engineering dCas9-based genetic circuits. PMID:26824432

  18. Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Beloglazova, Natalia; Petit, Pierre; Flick, Robert

    Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') single-stranded DNAs and RNAs, as well as 3'-flaps, splayed arms, and R-loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two boundmore » metal cations and together with site-directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo- and exonuclease activities.« less

  19. A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion

    PubMed Central

    Sampson, Timothy R.; Napier, Brooke A.; Schroeder, Max R.; Louwen, Rogier; Zhao, Jinshi; Chin, Chui-Yoke; Ratner, Hannah K.; Llewellyn, Anna C.; Jones, Crystal L.; Laroui, Hamed; Merlin, Didier; Zhou, Pei; Endtz, Hubert P.; Weiss, David S.

    2014-01-01

    Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals. PMID:25024199

  20. A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion.

    PubMed

    Sampson, Timothy R; Napier, Brooke A; Schroeder, Max R; Louwen, Rogier; Zhao, Jinshi; Chin, Chui-Yoke; Ratner, Hannah K; Llewellyn, Anna C; Jones, Crystal L; Laroui, Hamed; Merlin, Didier; Zhou, Pei; Endtz, Hubert P; Weiss, David S

    2014-07-29

    Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.

  1. CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

    PubMed

    Ehrke-Schulz, Eric; Schiwon, Maren; Leitner, Theo; Dávid, Stephan; Bergmann, Thorsten; Liu, Jing; Ehrhardt, Anja

    2017-12-07

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

  2. [The application of CRISPR/Cas9 genome editing technology in cancer research].

    PubMed

    Wang, Da-yong; Ma, Ning; Hui, Yang; Gao, Xu

    2016-01-01

    The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease) genome editing technology has become more and more popular in gene editing because of its simple design and easy operation. Using the CRISPR/Cas9 system, researchers can perform site-directed genome modification at the base level. Moreover, it has been widely used in genome editing in multiple species and related cancer research. In this review, we summarize the application of the CRISPR/Cas9 system in cancer research based on the latest research progresses as well as our understanding of cancer research and genome editing techniques.

  3. Probing the structural dynamics of the CRISPR-Cas9 RNA-guided DNA-cleavage system by coarse-grained modeling.

    PubMed

    Zheng, Wenjun

    2017-02-01

    In the adaptive immune systems of many bacteria and archaea, the Cas9 endonuclease forms a complex with specific guide/scaffold RNA to identify and cleave complementary target sequences in foreign DNA. This DNA targeting machinery has been exploited in numerous applications of genome editing and transcription control. However, the molecular mechanism of the Cas9 system is still obscure. Recently, high-resolution structures have been solved for Cas9 in different structural forms (e.g., unbound forms, RNA-bound binary complexes, and RNA-DNA-bound tertiary complexes, corresponding to an inactive state, a pre-target-bound state, and a cleavage-competent or product state), which offered key structural insights to the Cas9 mechanism. To further probe the structural dynamics of Cas9 interacting with RNA and DNA at the amino-acid level of details, we have performed systematic coarse-grained modeling using an elastic network model and related analyses. Our normal mode analysis predicted a few key modes of collective motions that capture the observed conformational changes featuring large domain motions triggered by binding of RNA and DNA. Our flexibility analysis identified specific regions with high or low flexibility that coincide with key functional sites (such as DNA/RNA-binding sites, nuclease cleavage sites, and key hinges). We also identified a small set of hotspot residues that control the energetics of functional motions, which overlap with known functional sites and offer promising targets for future mutagenesis efforts to improve the specificity of Cas9. Finally, we modeled the conformational transitions of Cas9 from the unbound form to the binary complex and then the tertiary complex, and predicted a distinct sequence of domain motions. In sum, our findings have offered rich structural and dynamic details relevant to the Cas9 machinery, and will guide future investigation and engineering of the Cas9 systems. Proteins 2017; 85:342-353. © 2016 Wiley Periodicals

  4. [Advances in CRISPR-Cas-mediated genome editing system in plants].

    PubMed

    Wang, Chun; Wang, Kejian

    2017-10-25

    Targeted genome editing technology is an important tool to study the function of genes and to modify organisms at the genetic level. Recently, CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) system has emerged as an efficient tool for specific genome editing in animals and plants. CRISPR-Cas system uses CRISPR-associated endonuclease and a guide RNA to generate double-strand breaks at the target DNA site, subsequently leading to genetic modifications. CRISPR-Cas system has received widespread attention for manipulating the genomes with simple, easy and high specificity. This review summarizes recent advances of diverse applications of the CRISPR-Cas toolkit in plant research and crop breeding, including expanding the range of genome editing, precise editing of a target base, and efficient DNA-free genome editing technology. This review also discusses the potential challenges and application prospect in the future, and provides a useful reference for researchers who are interested in this field.

  5. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Carte, Jason; Wang, Ruiying; Li, Hong

    An RNA-based gene silencing pathway that protects bacteria and archaea from viruses and other genome invaders is hypothesized to arise from guide RNAs encoded by CRISPR loci and proteins encoded by the cas genes. CRISPR loci contain multiple short invader-derived sequences separated by short repeats. The presence of virus-specific sequences within CRISPR loci of prokaryotic genomes confers resistance against corresponding viruses. The CRISPR loci are transcribed as long RNAs that must be processed to smaller guide RNAs. Here we identified Pyrococcus furiosus Cas6 as a novel endoribonuclease that cleaves CRISPR RNAs within the repeat sequences to release individual invader targetingmore » RNAs. Cas6 interacts with a specific sequence motif in the 5{prime} region of the CRISPR repeat element and cleaves at a defined site within the 3{prime} region of the repeat. The 1.8 angstrom crystal structure of the enzyme reveals two ferredoxin-like folds that are also found in other RNA-binding proteins. The predicted active site of the enzyme is similar to that of tRNA splicing endonucleases, and concordantly, Cas6 activity is metal-independent. cas6 is one of the most widely distributed CRISPR-associated genes. Our findings indicate that Cas6 functions in the generation of CRISPR-derived guide RNAs in numerous bacteria and archaea.« less

  6. Remedial action plan and site design for stabilization of the inactive uranium mill tailings sites at Rifle, Colorado

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Not Available

    1992-02-01

    This appendix assesses the present conditions and data gathered about the two inactive uranium mill tailings sites near Rifle, Colorado, and the designated disposal site six miles north of Rifle in the area of Estes Gulch. It consolidates available engineering, radiological, geotechnical, hydrological, meteorological, and other information pertinent to the design of the Remedial Action Plan (RAP). The data characterize conditions at the mill, tailings, and disposal site so that the Remedial Action Contractor (RAC) may complete final designs for the remedial actions.

  7. Closure Report for Corrective Action Unit 408: Bomblet Target Area Tonopah Test Range (TTR), Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mark Krauss

    2010-09-01

    This Closure Report (CR) presents information supporting the closure of Corrective Action Unit (CAU) 408: Bomblet Target Area (TTR), Tonopah Test Range, Nevada. This CR complies with the requirements of the Federal Facility Agreement and Consent Order that was agreed to by the State of Nevada; U.S. Department of Energy (DOE), Environmental Management; U.S. Department of Defense; and DOE, Legacy Management. Corrective Action Unit 408 is located at the Tonopah Test Range, Nevada, and consists of Corrective Action Site (CAS) TA-55-002-TAB2, Bomblet Target Areas. This CAS includes the following seven target areas: • Mid Target • Flightline Bomblet Location •more » Strategic Air Command (SAC) Target Location 1 • SAC Target Location 2 • South Antelope Lake • Tomahawk Location 1 • Tomahawk Location 2 The purpose of this CR is to provide documentation supporting the completed corrective actions and data confirming that the closure objectives for the CAS within CAU 408 were met. To achieve this, the following actions were performed: • Review the current site conditions, including the concentration and extent of contamination. • Implement any corrective actions necessary to protect human health and the environment. • Properly dispose of corrective action and investigation wastes. • Document Notice of Completion and closure of CAU 408 issued by the Nevada Division of Environmental Protection. From July 2009 through August 2010, closure activities were performed as set forth in the Streamlined Approach for Environmental Restoration Plan for CAU 408: Bomblet Target Area, Tonopah Test Range (TTR), Nevada. The purposes of the activities as defined during the data quality objectives process were as follows: • Identify and remove munitions of explosive concern (MEC) associated with DOE activities. • Investigate potential disposal pit locations. • Remove depleted uranium-contaminated fragments and soil. • Determine whether contaminants of concern

  8. Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field.

    PubMed

    He, Zhi-Yao; Men, Ke; Qin, Zhou; Yang, Yang; Xu, Ting; Wei, Yu-Quan

    2017-05-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs (sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies. Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.

  9. CRISPR/Cas9-Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development.

    PubMed

    Okoli, Arinze; Okeke, Malachy I; Tryland, Morten; Moens, Ugo

    2018-01-22

    The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them.

  10. Molecular basis for HEF1/NEDD9/Cas-L action as a multifunctional coordinator of invasion, apoptosis and cell cycle

    PubMed Central

    Singh, Mahendra K.; Cowell, Lauren; Seo, Sachiko; O’Neill, Geraldine M.; Golemis, Erica A.

    2007-01-01

    Upregulation of the scaffolding protein HEF1, also known as NEDD9 and Cas-L, has recently been identified as a pro-metastatic stimulus in a number of different solid tumors, and has also been strongly associated with pathogenesis of BCR-Abl-dependent tumors. As the evidence mounts for HEF1/NEDD9/Cas-L as a key player in metastatic cancer, it is timely to review the molecular regulation of HEF1/NEDD9/Cas-L. Most of the mortality associated with cancer arises from uncontrolled metastases, thus a better understanding of the properties of proteins specifically associated with promotion of this process may yield insights that improve cancer diagnosis and treatment. In this review, we summarize the extensive literature regarding HEF1/NEDD9/CAS-L expression and function in signaling relevant to cell attachment, migration, invasion; cell cycle; apoptosis; and oncogenic signal transduction. The complex function of HEF1/NEDD9/CAS-L revealed by this analysis leads us to propose a model in which alleviation of cell cycle checkpoints and acquired resistance to apoptosis is permissive for a HEF1/NEDD9/CAS-L-promoted pro-metastatic phenotype. PMID:17703068

  11. CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

    PubMed

    Xue, Zhaoyu; Wu, Menghua; Wen, Kejia; Ren, Menda; Long, Li; Zhang, Xuedi; Gao, Guanjun

    2014-09-05

    Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach. Copyright © 2014 Xue et al.

  12. Corrective Action Decision Document/Closure Report for Corrective Action Unit 570: Area 9 Yucca Flat Atmospheric Test Sites, Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    2013-11-01

    This Corrective Action Decision Document/Closure Report presents information supporting the closure of Corrective Action Unit (CAU) 570: Area 9 Yucca Flat Atmospheric Test Sites, Nevada National Security Site, Nevada. This complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; U.S. Department of Energy (DOE), Environmental Management; U.S. Department of Defense; and DOE, Legacy Management. The purpose of the CADD/CR is to provide justification and documentation supporting the recommendation that no further corrective action is needed.

  13. Closure Report for Corrective Action Unit 340: NTS Pesticide Release Sites Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    C. M. Obi

    The purpose of this report is to provide documentation of the completed corrective action and to provide data confirming the corrective action. The corrective action was performed in accordance with the approved Corrective Action Plan (CAP) (U.S. Department of Energy [DOE], 1999) and consisted of clean closure by excavation and disposal. The Area 15 Quonset Hut 15-11 was formerly used for storage of farm supplies including pesticides, herbicides, and fertilizers. The Area 23 Quonset Hut 800 was formerly used to clean pesticide and herbicide equipment. Steam-cleaning rinsate and sink drainage occasionally overflowed a sump into adjoining drainage ditches. One ditchmore » flows south and is referred to as the quonset hut ditch. The other ditch flows southeast and is referred to as the inner drainage ditch. The Area 23 Skid Huts were formerly used for storing and mixing pesticide and herbicide solutions. Excess solutions were released directly to the ground near the skid huts. The skid huts were moved to a nearby location prior to the site characterization performed in 1998 and reported in the Corrective Action Decision Document (CADD) (DOE, 1998). The vicinity and site plans of the Area 23 sites are shown in Figures 2 and 3, respectively.« less

  14. Cas9 versus Cas12a/Cpf1: Structure-function comparisons and implications for genome editing.

    PubMed

    Swarts, Daan C; Jinek, Martin

    2018-05-22

    Cas9 and Cas12a are multidomain CRISPR-associated nucleases that can be programmed with a guide RNA to bind and cleave complementary DNA targets. The guide RNA sequence can be varied, making these effector enzymes versatile tools for genome editing and gene regulation applications. While Cas9 is currently the best-characterized and most widely used nuclease for such purposes, Cas12a (previously named Cpf1) has recently emerged as an alternative for Cas9. Cas9 and Cas12a have distinct evolutionary origins and exhibit different structural architectures, resulting in distinct molecular mechanisms. Here we compare the structural and mechanistic features that distinguish Cas9 and Cas12a, and describe how these features modulate their activity. We discuss implications for genome editing, and how they may influence the choice of Cas9 or Cas12a for specific applications. Finally, we review recent studies in which Cas12a has been utilized as a genome editing tool. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes. © 2018 Wiley Periodicals, Inc.

  15. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases

    PubMed Central

    Lin, Lin; Liu, Yong; Xu, Fengping; Huang, Jinrong; Daugaard, Tina Fuglsang; Petersen, Trine Skov; Hansen, Bettina; Ye, Lingfei; Zhou, Qing; Fang, Fang; Yang, Ling; Li, Shengting; Fløe, Lasse; Jensen, Kristopher Torp; Shrock, Ellen; Chen, Fang; Yang, Huanming; Wang, Jian; Liu, Xin; Xu, Xun; Bolund, Lars; Nielsen, Anders Lade; Luo, Yonglun

    2018-01-01

    Abstract Background Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. Findings We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with

  16. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases.

    PubMed

    Lin, Lin; Liu, Yong; Xu, Fengping; Huang, Jinrong; Daugaard, Tina Fuglsang; Petersen, Trine Skov; Hansen, Bettina; Ye, Lingfei; Zhou, Qing; Fang, Fang; Yang, Ling; Li, Shengting; Fløe, Lasse; Jensen, Kristopher Torp; Shrock, Ellen; Chen, Fang; Yang, Huanming; Wang, Jian; Liu, Xin; Xu, Xun; Bolund, Lars; Nielsen, Anders Lade; Luo, Yonglun

    2018-03-01

    Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription. Our results prove that dCas

  17. The expanding footprint of CRISPR/Cas9 in the plant sciences

    USDA-ARS?s Scientific Manuscript database

    CRISPR/Cas9 has evolved and transformed the field of biology at an unprecedented pace. From the initial purpose of introducing a site specific mutation within a genome of choice, this technology has morphed into enabling a wide array of molecular applications, including site-specific transgene inser...

  18. CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.

    PubMed

    Modell, Joshua W; Jiang, Wenyan; Marraffini, Luciano A

    2017-04-06

    Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide protection against viral and plasmid infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host. These sequences, known as spacers, are transcribed into short CRISPR RNA guides that specify the cleavage site of Cas nucleases in the genome of the invader. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.

  19. CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

    PubMed

    Lemos, Brenda R; Kaplan, Adam C; Bae, Ji Eun; Ferrazzoli, Alexander E; Kuo, James; Anand, Ranjith P; Waterman, David P; Haber, James E

    2018-02-27

    Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

  20. Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

    NASA Astrophysics Data System (ADS)

    Zuo, Zhicheng; Liu, Jin

    2016-11-01

    The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

  1. CRISPR/Cas9-mediated targeted mutagenesis in grape

    PubMed Central

    Ban, Yusuke; Azuma, Akifumi; Onoue, Noriyuki; Moriguchi, Takaya; Yamamoto, Toshiya; Toki, Seiichi

    2017-01-01

    RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape—an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased. PMID:28542349

  2. Corrective Action Decision Document/Closure Report for Corrective Action Unit 569: Area 3 Yucca Flat Atmospheric Test Sites Nevada National Security Site, Nevada with ROTC 1, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sloop, Christy

    2013-04-01

    This Corrective Action Decision Document/Closure Report presents information supporting the closure of Corrective Action Unit (CAU) 569: Area 3 Yucca Flat Atmospheric Test Sites, Nevada National Security Site, Nevada. CAU 569 comprises the following nine corrective action sites (CASs): • 03-23-09, T-3 Contamination Area • 03-23-10, T-3A Contamination Area • 03-23-11, T-3B Contamination Area • 03-23-12, T-3S Contamination Area • 03-23-13, T-3T Contamination Area • 03-23-14, T-3V Contamination Area • 03-23-15, S-3G Contamination Area • 03-23-16, S-3H Contamination Area • 03-23-21, Pike Contamination Area The purpose of this Corrective Action Decision Document/Closure Report is to provide justification and documentation supportingmore » the recommendation that no further corrective action is needed for CAU 569 based on the implementation of the corrective actions listed in Table ES-2.« less

  3. Addendum to the Closure Report for Corrective Action Unit 165: Area 25 and 26 Dry Well and Washdown Areas, Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Krauss, Mark J

    This document constitutes an addendum to the Closure Report for Corrective Action Unit 165: Area 25 and 26 Dry Well and Washdown Areas, Nevada Test Site, Nevada as described in the document Recommendations and Justifications To Remove Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Field Office Federal Facility Agreement and Consent Order dated September 2013. The Use Restriction Removal document was approved by the Nevada Division of Environmental Protection on October 16, 2013. The approval of the UR Removal document constituted approval of each of the recommended UR removals. In conformance with themore » UR Removal document, this addendum consists of: This page that refers the reader to the UR Removal document for additional information The cover, title, and signature pages of the UR Removal document The NDEP approval letter The corresponding section of the UR Removal document This addendum provides the documentation justifying the cancellation of the UR for CAS 25-20-01, Lab Drain Dry Well. This UR was established as part of FFACO corrective actions and was based on the presence of tetrachloroethene contamination at concentrations greater than the action level established at the time of the initial investigation. Although total petroleum hydrocarbon diesel-range organics contamination at concentrations greater than the NDEP action level was present at the site, no hazardous constituents of TPH-DRO exceeded the U.S. Environmental Protection Agency (EPA) Region 9 preliminary remediation goals established at the time of the initial investigation.« less

  4. A genomic island in Vibrio cholerae with VPI-1 site-specific recombination characteristics contains CRISPR-Cas and type VI secretion modules

    PubMed Central

    Labbate, Maurizio; Orata, Fabini D.; Petty, Nicola K.; Jayatilleke, Nathasha D.; King, William L.; Kirchberger, Paul C.; Allen, Chris; Mann, Gulay; Mutreja, Ankur; Thomson, Nicholas R.; Boucher, Yan; Charles, Ian G.

    2016-01-01

    Cholera is a devastating diarrhoeal disease caused by certain strains of serogroup O1/O139 Vibrio cholerae. Mobile genetic elements such as genomic islands (GIs) have been pivotal in the evolution of O1/O139 V. cholerae. Perhaps the most important GI involved in cholera disease is the V. cholerae pathogenicity island 1 (VPI-1). This GI contains the toxin-coregulated pilus (TCP) gene cluster that is necessary for colonization of the human intestine as well as being the receptor for infection by the cholera-toxin bearing CTX phage. In this study, we report a GI (designated GIVchS12) from a non-O1/O139 strain of V. cholerae that is present in the same chromosomal location as VPI-1, contains an integrase gene with 94% nucleotide and 100% protein identity to the VPI-1 integrase, and attachment (att) sites 100% identical to those found in VPI-1. However, instead of TCP and the other accessory genes present in VPI-1, GIVchS12 contains a CRISPR-Cas element and a type VI secretion system (T6SS). GIs similar to GIVchS12 were identified in other V. cholerae genomes, also containing CRISPR-Cas elements and/or T6SS’s. This study highlights the diversity of GIs circulating in natural V. cholerae populations and identifies GIs with VPI-1 recombination characteristics as a propagator of CRISPR-Cas and T6SS modules. PMID:27845364

  5. Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting.

    PubMed

    Aguirre, Andrew J; Meyers, Robin M; Weir, Barbara A; Vazquez, Francisca; Zhang, Cheng-Zhong; Ben-David, Uri; Cook, April; Ha, Gavin; Harrington, William F; Doshi, Mihir B; Kost-Alimova, Maria; Gill, Stanley; Xu, Han; Ali, Levi D; Jiang, Guozhi; Pantel, Sasha; Lee, Yenarae; Goodale, Amy; Cherniack, Andrew D; Oh, Coyin; Kryukov, Gregory; Cowley, Glenn S; Garraway, Levi A; Stegmaier, Kimberly; Roberts, Charles W; Golub, Todd R; Meyerson, Matthew; Root, David E; Tsherniak, Aviad; Hahn, William C

    2016-08-01

    The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest. By examining single-guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR/Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR/Cas9 elicits a gene-independent antiproliferative cell response. This effect has important practical implications for the interpretation of CRISPR/Cas9 screening data and confounds the use of this technology for the identification of essential genes in amplified regions. We found that the number of CRISPR/Cas9-induced DNA breaks dictates a gene-independent antiproliferative response in cells. These observations have practical implications for using CRISPR/Cas9 to interrogate cancer gene function and illustrate that cancer cells are highly sensitive to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Cancer Discov; 6(8); 914-29. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Munoz et al., p. 900This article is highlighted in the In This Issue feature, p. 803. 2016 American Association for Cancer Research.

  6. ISC, a Novel Group of Bacterial and Archaeal DNA Transposons That Encode Cas9 Homologs

    PubMed Central

    Kapitonov, Vladimir V.; Makarova, Kira S.

    2015-01-01

    ABSTRACT Bacterial genomes encode numerous homologs of Cas9, the effector protein of the type II CRISPR-Cas systems. The homology region includes the arginine-rich helix and the HNH nuclease domain that is inserted into the RuvC-like nuclease domain. These genes, however, are not linked to cas genes or CRISPR. Here, we show that Cas9 homologs represent a distinct group of nonautonomous transposons, which we denote ISC (insertion sequences Cas9-like). We identify many diverse families of full-length ISC transposons and demonstrate that their terminal sequences (particularly 3′ termini) are similar to those of IS605 superfamily transposons that are mobilized by the Y1 tyrosine transposase encoded by the TnpA gene and often also encode the TnpB protein containing the RuvC-like endonuclease domain. The terminal regions of the ISC and IS605 transposons contain palindromic structures that are likely recognized by the Y1 transposase. The transposons from these two groups are inserted either exactly in the middle or upstream of specific 4-bp target sites, without target site duplication. We also identify autonomous ISC transposons that encode TnpA-like Y1 transposases. Thus, the nonautonomous ISC transposons could be mobilized in trans either by Y1 transposases of other, autonomous ISC transposons or by Y1 transposases of the more abundant IS605 transposons. These findings imply an evolutionary scenario in which the ISC transposons evolved from IS605 family transposons, possibly via insertion of a mobile group II intron encoding the HNH domain, and Cas9 subsequently evolved via immobilization of an ISC transposon. IMPORTANCE Cas9 endonucleases, the effectors of type II CRISPR-Cas systems, represent the new generation of genome-engineering tools. Here, we describe in detail a novel family of transposable elements that encode the likely ancestors of Cas9 and outline the evolutionary scenario connecting different varieties of these transposons and Cas9. PMID:26712934

  7. Nuclear facility decommissioning and site remedial actions. Volume 6. A selected bibliography

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Owen, P.T.; Michelson, D.C.; Knox, N.P.

    1985-09-01

    This bibliography of 683 references with abstracts on the subject of nuclear facility decommissioning, uranium mill tailings management, and site remedial actions is the sixth in a series of annual reports prepared for the US Department of Energy's Remedial Action Programs. Foreign as well as domestic literature of all types - technical reports, progress reports, journal articles, conference papers, symposium proceedings, theses, books, patents, legislation, and research project descriptions - has been included. The bibliography contains scientific (basic research as well as applied technology), economic, regulatory, and legal literature pertinent to the US Department of Energy's remedial action program. Majormore » chapters are: (1) Surplus Facilities Management Program; (2) Nuclear Facilities Decommissioning; (3) Formerly Utilized Sites Remedial Action Program; (4) Facilities Contaminated with Natural Radioactivity; (5) Uranium Mill Tailings Remedial Action Program; (6) Grand Junction Remedial Action Program; (7) Uranium Mill Tailings Management; (8) Technical Measurements Center; and (9) General Remedial Action Program Studies. Chapter sections for chapters 1, 2, 5, and 7 include Design, Planning, and Regulations; Environmental Studies and Site Surveys; Health, Safety, and Biomedical Studies; Decontamination Studies; Dismantlement and Demolition; Site Stabilization and Reclamation; Waste Disposal; Remedial Action Experience; and General Studies. The references within each chapter or section are arranged alphabetically by leading author. References having no individual author are arranged by corporate affiliation or by publication description.« less

  8. CRISPR/Cas9—Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development

    PubMed Central

    Okoli, Arinze; Okeke, Malachy I.; Tryland, Morten; Moens, Ugo

    2018-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them. PMID:29361752

  9. Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis.

    PubMed

    Rousseau, Beth A; Hou, Zhonggang; Gramelspacher, Max J; Zhang, Yan

    2018-03-01

    The microbial CRISPR systems enable adaptive defense against mobile elements and also provide formidable tools for genome engineering. The Cas9 proteins are type II CRISPR-associated, RNA-guided DNA endonucleases that identify double-stranded DNA targets by sequence complementarity and protospacer adjacent motif (PAM) recognition. Here we report that the type II-C CRISPR-Cas9 from Neisseria meningitidis (Nme) is capable of programmable, RNA-guided, site-specific cleavage and recognition of single-stranded RNA targets and that this ribonuclease activity is independent of the PAM sequence. We define the mechanistic feature and specificity constraint for RNA cleavage by NmeCas9 and also show that nuclease null dNmeCas9 binds to RNA target complementary to CRISPR RNA. Finally, we demonstrate that NmeCas9-catalyzed RNA cleavage can be blocked by three families of type II-C anti-CRISPR proteins. These results fundamentally expand the targeting capacities of CRISPR-Cas9 and highlight the potential utility of NmeCas9 as a single platform to target both RNA and DNA. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Remedial Action Plan and site design for stabilization of the inactive uranium mill tailings site at Durango, Colorado: Remedial action selection report. Revised final report

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Not Available

    1991-12-01

    The uranium mill tailings site near Durango, Colorado, was one of 24 inactive uranium mill sites designated to be remediated by the US Department of Energy (DOE) under the Uranium Mill Tailings Radiation Control Act of 1978 (UMTRCA). Part of the UMTRCA requires that the US Nuclear Regulatory Commission (NRC) concur with the DOE`s Remedial Action Plan (RAP) and certify that the remedial action conducted at the site complies with the standards promulgated by the US Environmental Protection Agency (EPA). Included in the RAP is this Remedial Action Selection Report (RAS), which has been developed to serve a two-fold purpose.more » First, it describes the activities that have been conducted by the DOE to accomplish remediation and long-term stabilization and control of the radioactive materials at the inactive uranium mill processing site near Durango, Colorado. Secondly, this document and the rest of the RAP, upon concurrence and execution by the DOE, the State of Colorado, and the NRC, become Appendix B of the Cooperative Agreement between the DOE and the State of Colorado.« less

  11. Off-target Effects in CRISPR/Cas9-mediated Genome Engineering

    PubMed Central

    Zhang, Xiao-Hui; Tee, Louis Y; Wang, Xiao-Gang; Huang, Qun-Shan; Yang, Shi-Hua

    2015-01-01

    CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)—RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site—is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype–phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology. PMID:26575098

  12. Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting.

    PubMed

    Chen, Fuqiang; Ding, Xiao; Feng, Yongmei; Seebeck, Timothy; Jiang, Yanfang; Davis, Gregory D

    2017-04-07

    Bacterial CRISPR-Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR-Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR-Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification.

  13. Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting

    PubMed Central

    Chen, Fuqiang; Ding, Xiao; Feng, Yongmei; Seebeck, Timothy; Jiang, Yanfang; Davis, Gregory D.

    2017-01-01

    Bacterial CRISPR–Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR–Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR–Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification. PMID:28387220

  14. Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells.

    PubMed

    Mercx, Sébastien; Tollet, Jérémie; Magy, Bertrand; Navarre, Catherine; Boutry, Marc

    2016-01-01

    Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells.

  15. Molecular Mechanisms of RNA-Targeting by Cas13-containing Type VI CRISPR-Cas Systems.

    PubMed

    O'Connell, Mitchell

    2018-06-22

    Prokaryotic adaptive immune systems use CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR associated (Cas) proteins for RNA-guided cleavage of foreign genetic elements. The focus of this review, Type VI CRISPR-Cas systems, include a single protein known as Cas13 (formerly C2c2), that when assembled with a crRNA forms a crRNA-guided RNA-targeting effector complex. Type VI CRISPR-Cas systems can be divided into four subtypes (A-D) based on Cas13 phylogeny. All Cas13 proteins studied to date possess two enzymatically distinct ribonuclease activities that are required for optimal interference. One RNase is responsible for pre-crRNA processing to form mature Type VI interference complexes, while the other RNase activity provided by the two HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) domains, is required for degradation of target RNA during viral interference. In this review, I will compare and contrast what is known about the molecular architecture and behavior of Type VI (A-D) CRISPR-Cas13 interference complexes, how this allows them to carry out their RNA-targeting function, how Type VI accessory proteins are able to modulate Cas13 activity, and how together all of these features have led to the rapid development of a range of RNA-targeting applications. Throughout I will also discuss some of the outstanding questions regarding Cas13's molecular behavior, and its role in bacterial adaptive immunity and RNA-targeting applications. Copyright © 2018. Published by Elsevier Ltd.

  16. CRISPR-Cas and Contact-Dependent Secretion Systems Present on Excisable Pathogenicity Islands with Conserved Recombination Modules.

    PubMed

    Carpenter, Megan R; Kalburge, Sai S; Borowski, Joseph D; Peters, Molly C; Colwell, Rita R; Boyd, E Fidelma

    2017-05-15

    Pathogenicity islands (PAIs) are mobile integrated genetic elements that contain a diverse range of virulence factors. PAIs integrate into the host chromosome at a tRNA locus that contains their specific bacterial attachment site, attB , via integrase-mediated site-specific recombination generating attL and attR sites. We identified conserved recombination modules (integrases and att sites) previously described in choleragenic Vibrio cholerae PAIs but with novel cargo genes. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins) and a type VI secretion system (T6SS) gene cluster were identified at the Vibrio pathogenicity island 1 (VPI-1) insertion site in 19 V. cholerae strains and contained the same recombination module. Two divergent type I-F CRISPR-Cas systems were identified, which differed in Cas protein homology and content. The CRISPR repeat sequence was identical among all V. cholerae strains, but the CRISPR spacer sequences and the number of spacers varied. In silico analysis suggests that the CRISPR-Cas systems were active against phages and plasmids. A type III secretion system (T3SS) was present in 12 V. cholerae strains on a 68-kb island inserted at the same tRNA-serine insertion site as VPI-2 and contained the same recombination module. Bioinformatics analysis showed that two divergent T3SSs exist among the strains examined. Both the CRISPR and T3SS islands excised site specifically from the bacterial chromosome as complete units, and the cognate integrases were essential for this excision. These data demonstrated that identical recombination modules that catalyze integration and excision from the chromosome can acquire diverse cargo genes, signifying a novel method of acquisition for both CRISPR-Cas systems and T3SSs. IMPORTANCE This work demonstrated the presence of CRISPR-Cas systems and T3SSs on PAIs. Our work showed that similar recombination modules can associate with different cargo genes and

  17. The Self-Inactivating KamiCas9 System for the Editing of CNS Disease Genes.

    PubMed

    Merienne, Nicolas; Vachey, Gabriel; de Longprez, Lucie; Meunier, Cécile; Zimmer, Virginie; Perriard, Guillaume; Canales, Mathieu; Mathias, Amandine; Herrgott, Lucas; Beltraminelli, Tim; Maulet, Axelle; Dequesne, Thomas; Pythoud, Catherine; Rey, Maria; Pellerin, Luc; Brouillet, Emmanuel; Perrier, Anselme L; du Pasquier, Renaud; Déglon, Nicole

    2017-09-19

    Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntington's disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease. Sequencing of potential off-targets with the constitutive Cas9 system in differentiated human iPSC revealed a very low incidence with only one site above background level. This off-target frequency was significantly reduced with the KamiCas9 system. These results demonstrate the potential of the self-inactivating CRISPR/Cas9 editing for applications in the context of neurodegenerative diseases. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  18. The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells.

    PubMed

    Daer, René M; Cutts, Josh P; Brafman, David A; Haynes, Karmella A

    2017-03-17

    In order to efficiently edit eukaryotic genomes, it is critical to test the impact of chromatin dynamics on CRISPR/Cas9 function and develop strategies to adapt the system to eukaryotic contexts. So far, research has extensively characterized the relationship between the CRISPR endonuclease Cas9 and the composition of the RNA-DNA duplex that mediates the system's precision. Evidence suggests that chromatin modifications and DNA packaging can block eukaryotic genome editing by custom-built DNA endonucleases like Cas9; however, the underlying mechanism of Cas9 inhibition is unclear. Here, we demonstrate that closed, gene-silencing-associated chromatin is a mechanism for the interference of Cas9-mediated DNA editing. Our assays use a transgenic cell line with a drug-inducible switch to control chromatin states (open and closed) at a single genomic locus. We show that closed chromatin inhibits binding and editing at specific target sites and that artificial reversal of the silenced state restores editing efficiency. These results provide new insights to improve Cas9-mediated editing in human and other mammalian cells.

  19. Corrective Action Decision Document/Closure Report for Corrective Action Unit 105: Area 2 Yucca Flat Atmospheric Test Sites, Nevada National Security Site, Nevada, Revision 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    2014-01-01

    The purpose of this Corrective Action Decision Document/Closure Report is to provide justification and documentation supporting the recommendation that no further corrective action is needed for CAU 105 based on the implementation of the corrective actions. Corrective action investigation (CAI) activities were performed from October 22, 2012, through May 23, 2013, as set forth in the Corrective Action Investigation Plan for Corrective Action Unit 105: Area 2 Yucca Flat Atmospheric Test Sites; and in accordance with the Soils Activity Quality Assurance Plan, which establishes requirements, technical planning, and general quality practices.

  20. A Guide to Computational Tools and Design Strategies for Genome Editing Experiments in Zebrafish Using CRISPR/Cas9.

    PubMed

    Prykhozhij, Sergey V; Rajan, Vinothkumar; Berman, Jason N

    2016-02-01

    The development of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology for mainstream biotechnological use based on its discovery as an adaptive immune mechanism in bacteria has dramatically improved the ability of molecular biologists to modify genomes of model organisms. The zebrafish is highly amenable to applications of CRISPR/Cas9 for mutation generation and a variety of DNA insertions. Cas9 protein in complex with a guide RNA molecule recognizes where to cut the homologous DNA based on a short stretch of DNA termed the protospacer-adjacent motif (PAM). Rapid and efficient identification of target sites immediately preceding PAM sites, quantification of genomic occurrences of similar (off target) sites and predictions of cutting efficiency are some of the features where computational tools play critical roles in CRISPR/Cas9 applications. Given the rapid advent and development of this technology, it can be a challenge for researchers to remain up to date with all of the important technological developments in this field. We have contributed to the armamentarium of CRISPR/Cas9 bioinformatics tools and trained other researchers in the use of appropriate computational programs to develop suitable experimental strategies. Here we provide an in-depth guide on how to use CRISPR/Cas9 and other relevant computational tools at each step of a host of genome editing experimental strategies. We also provide detailed conceptual outlines of the steps involved in the design and execution of CRISPR/Cas9-based experimental strategies, such as generation of frameshift mutations, larger chromosomal deletions and inversions, homology-independent insertion of gene cassettes and homology-based knock-in of defined point mutations and larger gene constructs.

  1. Nuclear facility decommissioning and site remedial actions: A selected bibliography: Volume 8

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Owen, P.T.; Michelson, D.C.; Knox, N.P.

    1987-09-01

    The 553 abstracted references on nuclear facility decommissioning, uranium mill tailings management, and site remedial actions constitute the eighth in a series of reports. Foreign and domestic literature of all types - technical reports, progress reports, journal articles, symposia proceedings, theses, books, patents, legislation, and research project descriptions - has been included. The bibliography contains scientific, technical, economic, regulatory, and legal information pertinent to the US Department of energy's remedial action program. Major chapters are Surplus Facilities Management Program, Nuclear Facilities Decommissioning, Formerly Utilized Sites Remedial Action Program, Facilities Contaminated with Naturally Occurring Radionuclides, Uranium Mill Tailings Remedial Action Program,more » Uranium Mill Tailings Management, Technical Measurements Center, and General Remedial Action Program Studies. Chapter sections for chapters 1, 2, 5, and 6 include Design, Planning, and Regulations; Environmental Studies and Site Surveys; Health, Safety, and Biomedical Studies; Decontamination Studies; Dismantlement and Demolition; Site Stabilization and Reclamation; Waste Disposal; Remedial Action Experience; and General Studies. Within these categories, references are arranged alphabetically by first author. Those references having no individual author are listed by corporate affiliation or by publication description. Indexes are provided for author, corporate affiliation, title word, publication description, geographic location, and keywords. The appendix contains a list of frequently used acronyms and abbreviations.« less

  2. Nuclear facility decommissioning and site remedial actions: a selected bibliography

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Owen, P.T.; Knox, N.P.; Fielden, J.M.

    This bibliography contains 693 references with abstracts on the subject of nuclear facility decommissioning, uranium mill tailings management, and site remedial actions. Foreign, as well as domestic, literature of all types - technical reports, progress reports, journal articles, conference papers, symposium proceedings, theses, books, patents, legislation, and research project descriptions - has been included in this publication. The bibliography contains scientific (basic research as well as applied technology), economic, regulatory, and legal literature pertinent to the US Department of Energy's Remedial Action Program. Major chapters are Surplus Facilities Management Program, Nuclear Facilities Decommissioning, Formerly Utilized Sites Remedial Action Program, Uraniummore » Mill Tailings Remedial Action Program, Grand Junction Remedial Action Program, and Uranium Mill Tailings Management. Chapter sections for chapters 1 and 2 include: Design, Planning, and Regulations; Site Surveys; Decontamination Studies; Dismantlement and Demolition; Land Decontamination and Reclamation; Waste Disposal; and General Studies. The references within each chapter are arranged alphabetically by leading author. References having no individual author are arranged by corporate author or by title. Indexes are provided for (1) author; (2) corporate affiliation; (3) title; (4) publication description; (5) geographic location; and (6) keywords. An appendix of 202 bibliographic references without abstracts or indexes has been included in this bibliography. This appendix represents literature identified but not abstracted due to time constraints.« less

  3. Corrective action investigation plan for CAU Number 453: Area 9 Landfill, Tonopah Test Range

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    This Corrective Action Investigation Plan (CAIP) contains the environmental sample collection objectives and criteria for conducting site investigation activities at the Area 9 Landfill, Corrective Action Unit (CAU) 453/Corrective Action (CAS) 09-55-001-0952, which is located at the Tonopah Test Range (TTR). The TTR, included in the Nellis Air Force Range, is approximately 255 kilometers (140 miles) northwest of Las Vegas, Nevada. The Area 9 Landfill is located northwest of Area 9 on the TTR. The landfill cells associated with CAU 453 were excavated to receive waste generated from the daily operations conducted at Area 9 and from range cleanup whichmore » occurred after test activities.« less

  4. Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research

    DOE PAGES

    Liu, Degao; Hu, Rongbin; Palla, Kaitlin J.; ...

    2016-02-18

    Genome editing with site-specific nucleases has become a powerful tool for functional characterization of plant genes and genetic improvement of agricultural crops. Among the various site-specific nuclease-based technologies available for genome editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems have shown the greatest potential for rapid and efficient editing of genomes in plant species. Here, this article reviews the current status of application of CRISPR/Cas9 to plant genomics research, with a focus on loss-of-function and gain-of-function analysis of individual genes in the context of perennial plants and the potential application of CRISPR/Cas9 to perturbation ofmore » gene expression, as well as identification and analysis of gene modules as part of an accelerated domestication and synthetic biology effort.« less

  5. Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Degao; Hu, Rongbin; Palla, Kaitlin J.

    Genome editing with site-specific nucleases has become a powerful tool for functional characterization of plant genes and genetic improvement of agricultural crops. Among the various site-specific nuclease-based technologies available for genome editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems have shown the greatest potential for rapid and efficient editing of genomes in plant species. Here, this article reviews the current status of application of CRISPR/Cas9 to plant genomics research, with a focus on loss-of-function and gain-of-function analysis of individual genes in the context of perennial plants and the potential application of CRISPR/Cas9 to perturbation ofmore » gene expression, as well as identification and analysis of gene modules as part of an accelerated domestication and synthetic biology effort.« less

  6. Genome editing: the road of CRISPR/Cas9 from bench to clinic

    PubMed Central

    Eid, Ayman; Mahfouz, Magdy M

    2016-01-01

    Molecular scissors engineered for site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes and could improve our understanding of the molecular underpinnings of disease states and facilitate novel therapeutic applications. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9). The CRISPR/Cas9 system's simplicity, facile engineering and amenability to multiplexing make it the system of choice for many applications. CRISPR/Cas9 has been used to generate disease models to study genetic diseases. Improvements are urgently needed for various aspects of the CRISPR/Cas9 system, including the system's precision, delivery and control over the outcome of the repair process. Here, we discuss the current status of genome engineering and its implications for the future of biological research and gene therapy. PMID:27741224

  7. Genome editing: the road of CRISPR/Cas9 from bench to clinic.

    PubMed

    Eid, Ayman; Mahfouz, Magdy M

    2016-10-14

    Molecular scissors engineered for site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes and could improve our understanding of the molecular underpinnings of disease states and facilitate novel therapeutic applications. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9). The CRISPR/Cas9 system's simplicity, facile engineering and amenability to multiplexing make it the system of choice for many applications. CRISPR/Cas9 has been used to generate disease models to study genetic diseases. Improvements are urgently needed for various aspects of the CRISPR/Cas9 system, including the system's precision, delivery and control over the outcome of the repair process. Here, we discuss the current status of genome engineering and its implications for the future of biological research and gene therapy.

  8. Nuclear facility decommissioning and site remedial actions: A selected bibliography, volume 9

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Owen, P.T.; Knox, N.P.; Michelson, D.C.

    1988-09-01

    The 604 abstracted references on nuclear facility decommissioning, uranium mill tailings management, and site remedial actions constitute the ninth in a series of reports prepared annually for the US Department of Energy's Remedial Action Programs. Foreign and domestic literature of all types--technical reports, progress reports, journal articles, symposia proceedings, theses, books, patents, legislation, and research project descriptions--has been included. The bibliography contains scientific, technical, economic, regulatory, and legal information pertinent to the US Department of Energy's remedial action programs. Major sections are (1) Surplus Facilities Management Program, (2) Nuclear Facilities Decommissioning, (3) Formerly Utilized Sites Remedial Action Program, (4) Facilitiesmore » Contaminated with Naturally Occurring Radionuclides, (5) Uranium Mill Tailings Remedial Action Program, (6) Uranium Mill Tailings Management, (7) Technical Measurements Center, and (8) General Remedial Action Program Studies. Subsections for sections 1, 2, 5, and 6 include: Design, Planning, and Regulations; Environmental Studies and Site Surveys; Health, Safety, and Biomedical Studies; Decontamination Studies; Dismantlement and Demolition; Site Stabilization and Reclamation; Waste Disposal; Remedial Action Experience; and General Studies. Within these categories, references are arranged alphabetically by first author. Those references having no individual author are listed by corporate affiliation or by publication description. Indexes are provided for author, corporate affiliation, title word, publication description, geographic location, and keywords. This report is a product of the Remedial Action Program Information Center (RAPIC), which selects and analyzes information on remedial actions and relevant radioactive waste management technologies. RAPIC staff and resources are available to meet a variety of information needs. Contact the center at (615) 576-0568 or FTS 626

  9. Efficient CRISPR/Cas9-based genome editing in carrot cells.

    PubMed

    Klimek-Chodacka, Magdalena; Oleszkiewicz, Tomasz; Lowder, Levi G; Qi, Yiping; Baranski, Rafal

    2018-04-01

    The first report presenting successful and efficient carrot genome editing using CRISPR/Cas9 system. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) is a powerful genome editing tool that has been widely adopted in model organisms recently, but has not been used in carrot-a model species for in vitro culture studies and an important health-promoting crop grown worldwide. In this study, for the first time, we report application of the CRISPR/Cas9 system for efficient targeted mutagenesis of the carrot genome. Multiplexing CRISPR/Cas9 vectors expressing two single-guide RNA (gRNAs) targeting the carrot flavanone-3-hydroxylase (F3H) gene were tested for blockage of the anthocyanin biosynthesis in a model purple-colored callus using Agrobacterium-mediated genetic transformation. This approach allowed fast and visual comparison of three codon-optimized Cas9 genes and revealed that the most efficient one in generating F3H mutants was the Arabidopsis codon-optimized AteCas9 gene with up to 90% efficiency. Knockout of F3H gene resulted in the discoloration of calli, validating the functional role of this gene in the anthocyanin biosynthesis in carrot as well as providing a visual marker for screening successfully edited events. Most resulting mutations were small Indels, but long chromosome fragment deletions of 116-119 nt were also generated with simultaneous cleavage mediated by two gRNAs. The results demonstrate successful site-directed mutagenesis in carrot with CRISPR/Cas9 and the usefulness of a model callus culture to validate genome editing systems. Given that the carrot genome has been sequenced recently, our timely study sheds light on the promising application of genome editing tools for boosting basic and translational research in this important vegetable crop.

  10. Nuclear facility decommissioning and site remedial actions: a selected bibliography. Volume 5

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Owen, P.T.; Knox, N.P.; Chilton, B.D.

    1984-09-01

    This bibliography of 756 references with abstracts on the subject of nuclear facility decommissioning, uranium mill tailings management, and site remedial actions is the fifth in a series of annual reports prepared for the US Department of Energy, Division of Remedial Action Projects. Foreign as well as domestic literature of all types - technical reports, progress reports, journal articles, conference papers, symposium proceedings, theses, books, patents, legislation, and research project descriptions - has been included in this publication. The bibliography contains scientific (basic research as well as applied technology), economic, regulatory, and legal literature pertinent to the US Department ofmore » Energy's Remedial Action Program. Major chapters are: (1) Surplus Facilities Management Program; (2) Nuclear Facilities Decommissioning; (3) Formerly Utilized Sites Remedial Action Program; (4) Uranium Mill Tailings Remedial Action Program; (5) Grand Junction Remedial Action Program; (6) Uranium Mill Tailings Management; and (7) Technical Measurements Center. Chapter sections for chapters 1, 2, 4, and 6 include Design, Planning, and Regulations; Environmental Studies and Site Surveys; Decontamination Studies; Dismantlement and Demolition; Site Stabilization and Reclamation; Waste Disposal; Remedial Action Experience; and General Studies. The references within each chapter or section are arranged alphabetically by leading author. References having no individual author are arranged by corporate author or by title. Indexes are provided for the categories of author, corporate affiliation, title, publication description, geographic location, and keywords. The Appendix contains a list of frequently used acronyms.« less

  11. Generation and comparison of CRISPR-Cas9 and Cre-mediated genetically engineered mouse models of sarcoma

    PubMed Central

    Huang, Jianguo; Chen, Mark; Whitley, Melodi Javid; Kuo, Hsuan-Cheng; Xu, Eric S.; Walens, Andrea; Mowery, Yvonne M.; Van Mater, David; Eward, William C.; Cardona, Diana M.; Luo, Lixia; Ma, Yan; Lopez, Omar M.; Nelson, Christopher E.; Robinson-Hamm, Jacqueline N.; Reddy, Anupama; Dave, Sandeep S.; Gersbach, Charles A.; Dodd, Rebecca D.; Kirsch, David G.

    2017-01-01

    Genetically engineered mouse models that employ site-specific recombinase technology are important tools for cancer research but can be costly and time-consuming. The CRISPR-Cas9 system has been adapted to generate autochthonous tumours in mice, but how these tumours compare to tumours generated by conventional recombinase technology remains to be fully explored. Here we use CRISPR-Cas9 to generate multiple subtypes of primary sarcomas efficiently in wild type and genetically engineered mice. These data demonstrate that CRISPR-Cas9 can be used to generate multiple subtypes of soft tissue sarcomas in mice. Primary sarcomas generated with CRISPR-Cas9 and Cre recombinase technology had similar histology, growth kinetics, copy number variation and mutational load as assessed by whole exome sequencing. These results show that sarcomas generated with CRISPR-Cas9 technology are similar to sarcomas generated with conventional modelling techniques and suggest that CRISPR-Cas9 can be used to more rapidly generate genotypically and phenotypically similar cancers. PMID:28691711

  12. Induced mutation and epigenetics modification in plants for crop improvement by targeting CRISPR/Cas9 technology.

    PubMed

    Khan, Muhammad Hafeez Ullah; Khan, Shahid U; Muhammad, Ali; Hu, Limin; Yang, Yang; Fan, Chuchuan

    2018-06-01

    Clustered regularly interspaced palindromic repeats associated protein Cas9 (CRISPR-Cas9), originally an adaptive immunity system of prokaryotes, is revolutionizing genome editing technologies with minimal off-targets in the present era. The CRISPR/Cas9 is now highly emergent, advanced, and highly specific tool for genome engineering. The technology is widely used to animal and plant genomes to achieve desirable results. The present review will encompass how CRISPR-Cas9 is revealing its beneficial role in characterizing plant genetic functions, genomic rearrangement, how it advances the site-specific mutagenesis, and epigenetics modification in plants to improve the yield of field crops with minimal side-effects. The possible pitfalls of using and designing CRISPR-Cas9 for plant genome editing are also discussed for its more appropriate applications in plant biology. Therefore, CRISPR/Cas9 system has multiple benefits that mostly scientists select for genome editing in several biological systems. © 2017 Wiley Periodicals, Inc.

  13. Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.

    PubMed

    Xue, Zhaoyu; Ren, Mengda; Wu, Menghua; Dai, Junbiao; Rong, Yikang S; Gao, Guanjun

    2014-03-21

    Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor. Copyright © 2014 Xue et al.

  14. A Convenient Cas9-based Conditional Knockout Strategy for Simultaneously Targeting Multiple Genes in Mouse.

    PubMed

    Chen, Jiang; Du, Yinan; He, Xueyan; Huang, Xingxu; Shi, Yun S

    2017-03-31

    The most powerful way to probe protein function is to characterize the consequence of its deletion. Compared to conventional gene knockout (KO), conditional knockout (cKO) provides an advanced gene targeting strategy with which gene deletion can be performed in a spatially and temporally restricted manner. However, for most species that are amphiploid, the widely used Cre-flox conditional KO (cKO) system would need targeting loci in both alleles to be loxP flanked, which in practice, requires time and labor consuming breeding. This is considerably significant when one is dealing with multiple genes. CRISPR/Cas9 genome modulation system is advantaged in its capability in targeting multiple sites simultaneously. Here we propose a strategy that could achieve conditional KO of multiple genes in mouse with Cre recombinase dependent Cas9 expression. By transgenic construction of loxP-stop-loxP (LSL) controlled Cas9 (LSL-Cas9) together with sgRNAs targeting EGFP, we showed that the fluorescence molecule could be eliminated in a Cre-dependent manner. We further verified the efficacy of this novel strategy to target multiple sites by deleting c-Maf and MafB simultaneously in macrophages specifically. Compared to the traditional Cre-flox cKO strategy, this sgRNAs-LSL-Cas9 cKO system is simpler and faster, and would make conditional manipulation of multiple genes feasible.

  15. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

    DOE PAGES

    Schumann, Kathrin; Lin, Steven; Boyer, Eric; ...

    2015-07-27

    T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4 + T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs).more » Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 ( PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.« less

  16. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schumann, Kathrin; Lin, Steven; Boyer, Eric

    T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4 + T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs).more » Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 ( PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.« less

  17. CRISPR-Cas9 systems: versatile cancer modelling platforms and promising therapeutic strategies.

    PubMed

    Wen, Wan-Shun; Yuan, Zhi-Min; Ma, Shi-Jie; Xu, Jiang; Yuan, Dong-Tang

    2016-03-15

    The RNA-guided nuclease CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9) and its variants such as nickase Cas9, dead Cas9, guide RNA scaffolds and RNA-targeting Cas9 are convenient and versatile platforms for site-specific genome editing and epigenome modulation. They are easy-to-use, simple-to-design and capable of targeting multiple loci simultaneously. Given that cancer develops from cumulative genetic and epigenetic alterations, CRISPR-Cas9 and its variants (hereafter referred to as CRISPR-Cas9 systems) hold extensive application potentials in cancer modeling and therapy. To date, they have already been applied to model oncogenic mutations in cell lines (e.g., Choi and Meyerson, Nat Commun 2014;5:3728) and in adult animals (e.g., Xue et al., Nature 2014;514:380-4), as well as to combat cancer by disabling oncogenic viruses (e.g., Hu et al., Biomed Res Int 2014;2014:612823) or by manipulating cancer genome (e.g., Liu et al., Nat Commun 2014;5:5393). Given the importance of epigenome and transcriptome in tumourigenesis, manipulation of cancer epigenome and transcriptome for cancer modeling and therapy is a promising area in the future. Whereas (epi)genetic modifications of cancer microenvironment with CRISPR-Cas9 systems for therapeutic purposes represent another promising area in cancer research. Herein, we introduce the functions and mechanisms of CRISPR-Cas9 systems in genome editing and epigenome modulation, retrospect their applications in cancer modelling and therapy, discuss limitations and possible solutions and propose future directions, in hope of providing concise and enlightening information for readers interested in this area. © 2015 UICC.

  18. Formerly Utilized Sites Remedial Action Program (FUSRAP) Hazelwood Interim Storage Site annual site environmental report. Calendar year 1985. [FUSRAP

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Not Available

    1986-04-01

    The Hazelwood Interim Storage Site (HISS) is presently used for the storage of low-level radioactively contaminated soils. Monitoring results show that the HISS is in compliance with DOE concentration guides and radiation protection standards. Derived Concentration Guides (DCGs) represent the concentrations of radionuclides in air or water that would limit the radiation dose to 100 mrem/y. The applicable limits have been revised since the 1984 environmental monitoring report was published. The limits applied in 1984 were based on a radiation protection standard of 500 mrem/y; the limits applied for 1985 are based on a standard of 100 mrem/y. The HISSmore » is part of the Formerly Utilized Sites Remedial Action Program (FUSRAP), a DOE program to decontaminate or otherwise control sites where low-level radioactive contamination remains from the early years of the nation's atomic energy program. To determine whether the site is in compliance with DOE standards, environmental measurements are expressed as percentages of the applicable DCG, while the calculated doses to the public are expressed as percentages of the applicable radiation protection standard. The monitoring program at the HISS measures uranium, radium, and thorium concentrations in surface water, groundwater, and sediment; radon gas concentrations in air; and external gamma radiation exposure rates. Potential radiation doses to the public are also calculated. The HISS was designated for remedial action under FUSRAP because radioactivity above applicable limits was found to exist at the site and its vicinity. Elevated levels of radiation still exist in areas where remedial action has not yet been completed.« less

  19. Corrrective action decision document for the Cactus Spring Waste Trenches (Corrective Action Unit No. 426). Revision No. 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    The Corrective Action Decision Document (CADD) for the Cactus Spring Waste Trenches (Corrective Action Unit [CAU] No. 426) has been prepared for the US Department of Energy`s (DOE) Nevada Environmental Restoration Project. This CADD has been developed to meet the requirements of the Federal Facility Agreement and Consent Order (FFACO) of 1996, stated in Appendix VI, {open_quotes}Corrective Action Strategy{close_quotes} (FFACO, 1996). The Cactus Spring Waste Trenches Corrective Action Site (CAS) No. RG-08-001-RG-CS is included in CAU No. 426 (also referred to as the {open_quotes}trenches{close_quotes}); it has been identified as one of three potential locations for buried, radioactively contaminated materials frommore » the Double Tracks Test. The trenches are located on the east flank of the Cactus Range in the eastern portion of the Cactus Spring Ranch at the Tonopah Test Range (TTR) in Nye County, Nevada, on the northern portion of Nellis Air Force Range. The TTR is approximately 225 kilometers (km) (140 miles [mi]) northwest of Las Vegas, Nevada, by air and approximately 56 km (35 mi) southeast of Tonopah, Nevada, by road. The trenches were dug for the purpose of receiving waste generated during Operation Roller Coaster, primarily the Double Tracks Test. This test, conducted in 1963, involved the use of live animals to assess the biological hazards associated with non-nuclear detonation of plutonium-bearing devices (i.e., inhalation uptake of plutonium aerosol). The CAS consists of four trenches that received solid waste and had an overall impacted area of approximately 36 meters (m) (120 feet [ft]) long x 24 m (80 ft) wide x 3 to 4.5 m (10 to 15 ft) deep. The average depressions at the trenches are approximately 0.3 m (1 ft) below land surface.« less

  20. A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease

    USDA-ARS?s Scientific Manuscript database

    The Cas9 endonuclease of the Type II-a clustered regularly interspersed short palindromic repeats (CRISPR), of Streptococcus pyogenes (SpCas9) has been adapted as a widely used tool for genome editing and genome engineering. Herein, we describe a gene encoding a novel Cas9 ortholog (BpsuCas9) and th...

  1. A RecET-assisted CRISPR-Cas9 genome editing in Corynebacterium glutamicum.

    PubMed

    Wang, Bo; Hu, Qitiao; Zhang, Yu; Shi, Ruilin; Chai, Xin; Liu, Zhe; Shang, Xiuling; Zhang, Yun; Wen, Tingyi

    2018-04-23

    Extensive modification of genome is an efficient manner to regulate the metabolic network for producing target metabolites or non-native products using Corynebacterium glutamicum as a cell factory. Genome editing approaches by means of homologous recombination and counter-selection markers are laborious and time consuming due to multiple round manipulations and low editing efficiencies. The current two-plasmid-based CRISPR-Cas9 editing methods generate false positives due to the potential instability of Cas9 on the plasmid, and require a high transformation efficiency for co-occurrence of two plasmids transformation. Here, we developed a RecET-assisted CRISPR-Cas9 genome editing method using a chromosome-borne Cas9-RecET and a single plasmid harboring sgRNA and repair templates. The inducible expression of chromosomal RecET promoted the frequencies of homologous recombination, and increased the efficiency for gene deletion. Due to the high transformation efficiency of a single plasmid, this method enabled 10- and 20-kb region deletion, 2.5-, 5.7- and 7.5-kb expression cassette insertion and precise site-specific mutation, suggesting a versatility of this method. Deletion of argR and farR regulators as well as site-directed mutation of argB and pgi genes generated the mutant capable of accumulating L-arginine, indicating the stability of chromosome-borne Cas9 for iterative genome editing. Using this method, the model-predicted target genes were modified to redirect metabolic flux towards 1,2-propanediol biosynthetic pathway. The final engineered strain produced 6.75 ± 0.46 g/L of 1,2-propanediol that is the highest titer reported in C. glutamicum. Furthermore, this method is available for Corynebacterium pekinense 1.563, suggesting its universal applicability in other Corynebacterium species. The RecET-assisted CRISPR-Cas9 genome editing method will facilitate engineering of metabolic networks for the synthesis of interested bio-based products from renewable

  2. [Detection of CRSPR-Cas system in Streptococcus thermophiles].

    PubMed

    Li, Wan; Liang, Hongzhang; Zhang, Danqing; Wang, Nana; Tang, Yaru; Li, Bailiang; Huo, Guicheng

    2016-04-14

    We aimed to detect the CRSPR-Cas system of six Streptococcus thermophilus. Bioinformatics method was used to predict CRSPR-Cas system of nine S. thermophilus that published in National Center for Biotechnology Information. Four primers were designed according to the flanking sequences of standard strains and the CRISPR-Cas system of six S. thermophilus have been detected by PCR method. S. thermophilus S4 had a Cas9 gene, others all had Cas9 gene, Cas10 gene and Cas9* gene. In addition, 79 and KLDS3.0207 still had Cas3 gene. Signature genes amplification of CRSPR-Cas system could predict the type of CRSPR-Cas system in unsequenced strains, these findings will help establish the foundation for the study of CRSPR-Cas system in lactic acid bacteria.

  3. Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

    PubMed Central

    Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

    2017-01-01

    The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA–DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA–DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs. PMID:28484024

  4. Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation.

    PubMed

    Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

    2017-05-23

    The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

  5. Second Line of Defense Virtual Private Network Guidance for Deployed and New CAS Systems

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Singh, Surya V.; Thronas, Aaron I.

    2010-01-01

    This paper discusses the importance of remote access via virtual private network (VPN) for the Second Line of Defense (SLD) Central Alarm System (CAS) sites, the requirements for maintaining secure channels while using VPN and implementation requirements for current and future sites.

  6. Building Cre Knockin Rat Lines Using CRISPR/Cas9.

    PubMed

    Ma, Yuanwu; Zhang, Lianfeng; Huang, Xingxu

    2017-01-01

    Conditional gene inactivation strategy helps researchers to study the gene functions that are critical in embryogenesis or in defined tissues of adulthood. The Cre/loxP system is widely used for conditional gene inactivation/activation in cells or organisms. Cre knockin animal lines are essential for gene expression or inactivation in a spatially and temporally restricted manner. However, to generate a Cre knockin line by traditional approach is laborious. Recently, the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) has been proven as a simple and efficient genome-editing tool. We have used CRISPR/Cas9 system to generate rat strains that carry Cre genes in different targeted gene loci by direct delivery of gRNAs/Cas9/donors into fertilized eggs. Here, we described a stepwise procedure for the generation of Cre knockin rat, including target site selection, RNA preparation, the construction of the template donor, pronuclear injection, and the genotyping of precise Cre insertion in F 0 rats. Taken together, the establishment of Cre knockin line can be achieved within 6 weeks.

  7. 2013 Annual Summary Report for the Area 3 and Area 5 Radioactive Waste Management Sites at the Nevada National Security Site, Nye County, Nevada; Review of the Performance Assessments and Composite Analyses

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shott, Gregory

    2014-03-01

    The Maintenance Plan for the Performance Assessments and Composite Analyses for the Area 3 and Area 5 Radioactive Waste Management Sites at the Nevada Test Site (National Security Technologies, LLC 2007a) requires an annual review to assess the adequacy of the performance assessments (PAs) and composite analyses (CAs), with the results submitted to the U.S. Department of Energy (DOE) Office of Environmental Management. The Disposal Authorization Statements for the Area 3 and Area 5 Radioactive Waste Management Sites (RWMSs) also require that such reviews be made and that secondary or minor unresolved issues be tracked and addressed as part ofmore » the maintenance plan (DOE 1999a, 2000). The U.S. Department of Energy, National Nuclear Security Administration Nevada Field Office performed an annual review of the Area 3 and Area 5 RWMS PAs and CAs for fiscal year (FY) 2013. This annual summary report presents data and conclusions from the FY 2013 review, and determines the adequacy of the PAs and CAs. Operational factors (e.g., waste forms and containers, facility design, and waste receipts), closure plans, monitoring results, and research and development (R&D) activities were reviewed to determine the adequacy of the PAs. Likewise, the environmental restoration activities at the Nevada National Security Site (NNSS) relevant to the sources of residual radioactive material that are considered in the CAs, the land-use planning, and the results of the environmental monitoring and R&D activities were reviewed to determine the adequacy of the CAs. Important developments in FY 2013 include the following: • Development of a new Area 5 RWMS closure inventory estimate based on disposals through FY 2013 • Evaluation of new or revised waste streams by special analysis • Development of version 4.115 of the Area 5 RWMS GoldSim PA/CA model The Area 3 RWMS has been in inactive status since July 1, 2006, with the last shipment received in April 2006. The FY 2013 review of

  8. A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease.

    PubMed

    Ivanov, Yury V; Shariat, Nikki; Register, Karen B; Linz, Bodo; Rivera, Israel; Hu, Kai; Dudley, Edward G; Harvill, Eric T

    2015-10-26

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) are widely distributed among bacteria. These systems provide adaptive immunity against mobile genetic elements specified by the spacer sequences stored within the CRISPR. The CRISPR-Cas system has been identified using Basic Local Alignment Search Tool (BLAST) against other sequenced and annotated genomes and confirmed via CRISPRfinder program. Using Polymerase Chain Reactions (PCR) and Sanger DNA sequencing, we discovered CRISPRs in additional bacterial isolates of the same species of Bordetella. Transcriptional activity and processing of the CRISPR have been assessed via RT-PCR. Here we describe a novel Type II-C CRISPR and its associated genes-cas1, cas2, and cas9-in several isolates of a newly discovered Bordetella species. The CRISPR-cas locus, which is absent in all other Bordetella species, has a significantly lower GC-content than the genome-wide average, suggesting acquisition of this locus via horizontal gene transfer from a currently unknown source. The CRISPR array is transcribed and processed into mature CRISPR RNAs (crRNA), some of which have homology to prophages found in closely related species B. hinzii. Expression of the CRISPR-Cas system and processing of crRNAs with perfect homology to prophages present in closely related species, but absent in that containing this CRISPR-Cas system, suggest it provides protection against phage predation. The 3,117-bp cas9 endonuclease gene from this novel CRISPR-Cas system is 990 bp smaller than that of Streptococcus pyogenes, the 4,017-bp allele currently used for genome editing, and which may make it a useful tool in various CRISPR-Cas technologies.

  9. 2012 Annual Summary Report for the Area 3 and Area 5 Radioactive Waste Management Sites at the Nevada National Security Site, Nye County, Nevada: Review of the Performance Assessments and Composite Analyses

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shott, G.

    2013-03-18

    The Maintenance Plan for the Performance Assessments and Composite Analyses for the Area 3 and Area 5 Radioactive Waste Management Sites at the Nevada Test Site (National Security Technologies, LLC 2007a) requires an annual review to assess the adequacy of the performance assessments (PAs) and composite analyses (CAs), with the results submitted to the U.S. Department of Energy (DOE) Office of Environmental Management. The Disposal Authorization Statements for the Area 3 and Area 5 Radioactive Waste Management Sites (RWMSs) also require that such reviews be made and that secondary or minor unresolved issues be tracked and addressed as part ofmore » the maintenance plan (DOE 1999a, 2000). The U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office performed an annual review of the Area 3 and Area 5 RWMS PAs and CAs for fiscal year (FY) 2012. This annual summary report presents data and conclusions from the FY 2012 review, and determines the adequacy of the PAs and CAs. Operational factors (e.g., waste forms and containers, facility design, and waste receipts), closure plans, monitoring results, and research and development (R&D) activities were reviewed to determine the adequacy of the PAs. Likewise, the environmental restoration activities at the Nevada National Security Site (NNSS) relevant to the sources of residual radioactive material that are considered in the CAs, the land-use planning, and the results of the environmental monitoring and R&D activities were reviewed to determine the adequacy of the CAs. Important developments in FY 2012 include the following: Release of a special analysis for the Area 3 RWMS assessing the continuing validity of the PA and CA; Development of a new Area 5 RWMS closure inventory estimate based on disposals through FY 2012; Evaluation of new or revised waste streams by special analysis; and Development of version 4.114 of the Area 5 RWMS GoldSim PA model. The Area 3 RWMS has been in inactive

  10. Breaking-Cas—interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes

    PubMed Central

    Oliveros, Juan C.; Franch, Mònica; Tabas-Madrid, Daniel; San-León, David; Montoliu, Lluis; Cubas, Pilar; Pazos, Florencio

    2016-01-01

    The CRISPR/Cas technology is enabling targeted genome editing in multiple organisms with unprecedented accuracy and specificity by using RNA-guided nucleases. A critical point when planning a CRISPR/Cas experiment is the design of the guide RNA (gRNA), which directs the nuclease and associated machinery to the desired genomic location. This gRNA has to fulfil the requirements of the nuclease and lack homology with other genome sites that could lead to off-target effects. Here we introduce the Breaking-Cas system for the design of gRNAs for CRISPR/Cas experiments, including those based in the Cas9 nuclease as well as others recently introduced. The server has unique features not available in other tools, including the possibility of using all eukaryotic genomes available in ENSEMBL (currently around 700), placing variable PAM sequences at 5′ or 3′ and setting the guide RNA length and the scores per nucleotides. It can be freely accessed at: http://bioinfogp.cnb.csic.es/tools/breakingcas, and the code is available upon request. PMID:27166368

  11. Addendum to the Closure Report for Corrective Action Unit 271: Areas 25, 26, and 27 Septic Systems Nevada Test Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the August 2004, Closure Report for Corrective Action Unit 271, Areas 25, 26, and 27 Septic Systems as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. The approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modification document, this addendum consistsmore » of: • This cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the modification of the UR for CAS 27-05-02, Leachfield. This UR was established as part of a Federal Facility Agreement and Consent Order (FFACO) corrective action and is based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since this UR was established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, this UR was re-evaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006c). This re-evaluation consisted of comparing the original data (used to define the need for the UR) to risk-based final action levels (FALs) developed using the current Industrial Sites RBCA process. The re-evaluation resulted in a recommendation to modify the UR to an administrative UR. Administrative URs differ

  12. A Broad-Spectrum Inhibitor of CRISPR-Cas9.

    PubMed

    Harrington, Lucas B; Doxzen, Kevin W; Ma, Enbo; Liu, Jun-Jie; Knott, Gavin J; Edraki, Alireza; Garcia, Bianca; Amrani, Nadia; Chen, Janice S; Cofsky, Joshua C; Kranzusch, Philip J; Sontheimer, Erik J; Davidson, Alan R; Maxwell, Karen L; Doudna, Jennifer A

    2017-09-07

    CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. 32 CFR 37.570 - What must I do if a CAS-covered participant accounts differently for its own and the Federal...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Accounting Standard (CAS) 402. Noncompliance with CAS 402 is a potential issue only for a participant that... contractors with their ACOs (currently on the World Wide Web at http://alerts.dcmdw.dcma.mil/support, a site...

  14. 2010 Annual Summary Report for the Area 3 and Area 5 Radioactive Management Sites at the Nevada National Security Site, Nye County, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Management

    2011-03-01

    The U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office performed an annual review of the Area 3 and Area 5 Radioactive Waste Management Site (RWMS) Performance Assessments (PAs) and Composite Analyses (CAs) in fiscal year (FY) 2010. This annual summary report presents data and conclusions from the FY 2010 review, and determines the adequacy of the PAs and CAs. Operational factors (e.g., waste forms and containers, facility design, and waste receipts), closure plans, monitoring results, and research and development (R&D) activities were reviewed to determine the adequacy of the PAs. Likewise, the environmental restoration activities at themore » Nevada National Security Site (NNSS) (formerly the Nevada Test Site) relevant to the sources of residual radioactive material that are considered in the CAs, the land-use planning, and the results of the environmental monitoring and R&D activities were reviewed to determine the adequacy of the CAs.« less

  15. CRISPR/Cas9-Based Multiplex Genome Editing in Monocot and Dicot Plants.

    PubMed

    Ma, Xingliang; Liu, Yao-Guang

    2016-07-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome targeting system has been applied to a variety of organisms, including plants. Compared to other genome-targeting technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas9 system is easier to use and has much higher editing efficiency. In addition, multiple "single guide RNAs" (sgRNAs) with different target sequences can be designed to direct the Cas9 protein to multiple genomic sites for simultaneous multiplex editing. Here, we present a procedure for highly efficient multiplex genome targeting in monocot and dicot plants using a versatile and robust CRISPR/Cas9 vector system, emphasizing the construction of binary constructs with multiple sgRNA expression cassettes in one round of cloning using Golden Gate ligation. We also describe the genotyping of targeted mutations in transgenic plants by direct Sanger sequencing followed by decoding of superimposed sequencing chromatograms containing biallelic or heterozygous mutations using the Web-based tool DSDecode. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  16. Annotation and Classification of CRISPR-Cas Systems

    PubMed Central

    Makarova, Kira S.; Koonin, Eugene V.

    2018-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is a prokaryotic adaptive immune system that is represented in most archaea and many bacteria. Among the currently known prokaryotic defense systems, the CRISPR-Cas genomic loci show unprecedented complexity and diversity. Classification of CRISPR-Cas variants that would capture their evolutionary relationships to the maximum possible extent is essential for comparative genomic and functional characterization of this theoretically and practically important system of adaptive immunity. To this end, a multipronged approach has been developed that combines phylogenetic analysis of the conserved Cas proteins with comparison of gene repertoires and arrangements in CRISPR-Cas loci. This approach led to the current classification of CRISPR-Cas systems into three distinct types and ten subtypes for each of which signature genes have been identified. Comparative genomic analysis of the CRISPR-Cas systems in new archaeal and bacterial genomes performed over the 3 years elapsed since the development of this classification makes it clear that new types and subtypes of CRISPR-Cas need to be introduced. Moreover, this classification system captures only part of the complexity of CRISPR-Cas organization and evolution, due to the intrinsic modularity and evolutionary mobility of these immunity systems, resulting in numerous recombinant variants. Moreover, most of the cas genes evolve rapidly, complicating the family assignment for many Cas proteins and the use of family profiles for the recognition of CRISPR-Cas subtype signatures. Further progress in the comparative analysis of CRISPR-Cas systems requires integration of the most sensitive sequence comparison tools, protein structure comparison, and refined approaches for comparison of gene neighborhoods. PMID:25981466

  17. Annotation and Classification of CRISPR-Cas Systems.

    PubMed

    Makarova, Kira S; Koonin, Eugene V

    2015-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is a prokaryotic adaptive immune system that is represented in most archaea and many bacteria. Among the currently known prokaryotic defense systems, the CRISPR-Cas genomic loci show unprecedented complexity and diversity. Classification of CRISPR-Cas variants that would capture their evolutionary relationships to the maximum possible extent is essential for comparative genomic and functional characterization of this theoretically and practically important system of adaptive immunity. To this end, a multipronged approach has been developed that combines phylogenetic analysis of the conserved Cas proteins with comparison of gene repertoires and arrangements in CRISPR-Cas loci. This approach led to the current classification of CRISPR-Cas systems into three distinct types and ten subtypes for each of which signature genes have been identified. Comparative genomic analysis of the CRISPR-Cas systems in new archaeal and bacterial genomes performed over the 3 years elapsed since the development of this classification makes it clear that new types and subtypes of CRISPR-Cas need to be introduced. Moreover, this classification system captures only part of the complexity of CRISPR-Cas organization and evolution, due to the intrinsic modularity and evolutionary mobility of these immunity systems, resulting in numerous recombinant variants. Moreover, most of the cas genes evolve rapidly, complicating the family assignment for many Cas proteins and the use of family profiles for the recognition of CRISPR-Cas subtype signatures. Further progress in the comparative analysis of CRISPR-Cas systems requires integration of the most sensitive sequence comparison tools, protein structure comparison, and refined approaches for comparison of gene neighborhoods.

  18. Corrective Action Investigation Plan for Corrective Action Unit 528: Polychlorinated Biphenyls Contamination, Nevada Test Site, Nevada, Rev. 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office

    This Corrective Action Investigation Plan contains the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office's approach to collect the data necessary to evaluate corrective action alternatives appropriate for the closure of Corrective Action Unit (CAU) 528, Polychlorinated Biphenyls Contamination (PCBs), Nevada Test Site (NTS), Nevada, under the Federal Facility Agreement and Consent Order. Located in the southwestern portion of Area 25 on the NTS in Jackass Flats (adjacent to Test Cell C [TCC]), CAU 528 consists of Corrective Action Site 25-27-03, Polychlorinated Biphenyls Surface Contamination. Test Cell C was built to support the Nuclear Rocket Development Stationmore » (operational between 1959 and 1973) activities including conducting ground tests and static firings of nuclear engine reactors. Although CAU 528 was not considered as a direct potential source of PCBs and petroleum contamination, two potential sources of contamination have nevertheless been identified from an unknown source in concentrations that could potentially pose an unacceptable risk to human health and/or the environment. This CAU's close proximity to TCC prompted Shaw to collect surface soil samples, which have indicated the presence of PCBs extending throughout the area to the north, east, south, and even to the edge of the western boundary. Based on this information, more extensive field investigation activities are being planned, the results of which are to be used to support a defensible evaluation of corrective action alternatives in the corrective action decision document.« less

  19. Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants.

    PubMed

    Luo, Ming; Gilbert, Brian; Ayliffe, Michael

    2016-07-01

    Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described.

  20. Naturally occurring off-switches for CRISPR-Cas9

    PubMed Central

    Pawluk, April; Amrani, Nadia; Zhang, Yan; Garcia, Bianca; Hidalgo-Reyes, Yurima; Lee, Jooyoung; Edraki, Alireza; Shah, Megha; Sontheimer, Erik J.; Maxwell, Karen L.; Davidson, Alan R.

    2017-01-01

    Summary CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These “anti-CRISPRs” were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9), and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable “off-switches” for CRISPR-Cas9 activity, and provide a genetically-encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. PMID:27984730

  1. [Chromosomal large fragment deletion induced by CRISPR/Cas9 gene editing system].

    PubMed

    Cheng, L H; Liu, Y; Niu, T

    2017-05-14

    Objective: Using CRISPR-Cas9 gene editing technology to achieve a number of genes co-deletion on the same chromosome. Methods: CRISPR-Cas9 lentiviral plasmid that could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse 11B3 chromosome was constructed via molecular clone. HEK293T cells were transfected to package lentivirus of CRISPR or Cas9 cDNA, then mouse NIH3T3 cells were infected by lentivirus and genomic DNA of these cells was extracted. The deleted fragment was amplified by PCR, TA clone, Sanger sequencing and other techniques were used to confirm the deletion of Aloxe3-Alox12b-Alox8 cluster genes. Results: The CRISPR-Cas9 lentiviral plasmid, which could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes, was successfully constructed. Deletion of target chromosome fragment (Aloxe3-Alox12b-Alox8 cluster genes) was verified by PCR. The deletion of Aloxe3-Alox12b-Alox8 cluster genes was affirmed by TA clone, Sanger sequencing, and the breakpoint junctions of the CRISPR-Cas9 system mediate cutting events were accurately recombined, insertion mutation did not occur between two cleavage sites at all. Conclusion: Large fragment deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse chromosome 11B3 was successfully induced by CRISPR-Cas9 gene editing system.

  2. CRISPR-Cas: Adapting to change.

    PubMed

    Jackson, Simon A; McKenzie, Rebecca E; Fagerlund, Robert D; Kieper, Sebastian N; Fineran, Peter C; Brouns, Stan J J

    2017-04-07

    Bacteria and archaea are engaged in a constant arms race to defend against the ever-present threats of viruses and invasion by mobile genetic elements. The most flexible weapons in the prokaryotic defense arsenal are the CRISPR-Cas adaptive immune systems. These systems are capable of selective identification and neutralization of foreign DNA and/or RNA. CRISPR-Cas systems rely on stored genetic memories to facilitate target recognition. Thus, to keep pace with a changing pool of hostile invaders, the CRISPR memory banks must be regularly updated with new information through a process termed CRISPR adaptation. In this Review, we outline the recent advances in our understanding of the molecular mechanisms governing CRISPR adaptation. Specifically, the conserved protein machinery Cas1-Cas2 is the cornerstone of adaptive immunity in a range of diverse CRISPR-Cas systems. Copyright © 2017, American Association for the Advancement of Science.

  3. Closure Report for Corrective Action Unit 394: Areas 12, 18, and 29 Spill/Release Sites, Nevada Test Site, Nevada: Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office

    This Closure Report (CR) presents information supporting a closure recommendation for Corrective Action Unit (CAU) 394: Areas 12, 18, and 29 Spill/Release Sites, Nevada Test Site, Nevada, in compliance with the requirements of the Federal Facility Agreement and Consent Order. This CAU contains six Corrective Action Sites (CASs): 12-25-04, UST 12-16-2 Waste Oil Release; 18-25-01, 18-25-02, 18-25-03, Oil Spills; 18-25-04, Spill (Diesel Fuel); and 29-44-01, Fuel Spill, located within Areas 12, 18, and 29 on the Nevada Test Site. The purpose of this CR is to provide documentation supporting recommendations of no further action or closure in place for CASsmore » within CAU 394. Throughout late 2002 and early to mid 2003, closure activities were performed as set forth in the CAU 394 Streamlined Approach for Environmental Restoration Plan. The closure activities identified the nature and extent of contaminants of potential concern at the CASs, and provided sufficient information and data to complete appropriate corrective actions for the CASs. Soil in CASs 18-25-02 and 18-25-03 containing polychlorinated biphenyls exceeding the action levels established by the Nevada Administrative Code were removed for proper disposal. The soil remaining in these CASs containing petroleum hydrocarbons exceeding the action level were closed in place with use restrictions. Corrective Action Sites 18-25-04 required no further corrective action; closure in place is required at CASs 12-25-04, 18-25-01, 18-25-02, 18-25-03, and 29-44-01; and use restrictions are required at CASs 12-25-04, 18-25-01, 18-25-02, 18-25-03 and 29-44-01. In summary, no corrective action plan is required for CAU 394.« less

  4. New CRISPR-Cas systems from uncultivated microbes

    NASA Astrophysics Data System (ADS)

    Burstein, David; Harrington, Lucas B.; Strutt, Steven C.; Probst, Alexander J.; Anantharaman, Karthik; Thomas, Brian C.; Doudna, Jennifer A.; Banfield, Jillian F.

    2017-02-01

    CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.

  5. Mobile Genetic Elements and Evolution of CRISPR-Cas Systems: All the Way There and Back.

    PubMed

    Koonin, Eugene V; Makarova, Kira S

    2017-10-01

    The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) systems of bacterial and archaeal adaptive immunity show multifaceted evolutionary relationships with at least five classes of mobile genetic elements (MGE). First, the adaptation module of CRISPR-Cas that is responsible for the formation of the immune memory apparently evolved from a Casposon, a self-synthesizing transposon that employs the Cas1 protein as the integrase and might have brought additional cas genes to the emerging immunity loci. Second, a large subset of type III CRISPR-Cas systems recruited a reverse transcriptase from a Group II intron, providing for spacer acquisition from RNA. Third, effector nucleases of Class 2 CRISPR-Cas systems that are responsible for the recognition and cleavage of the target DNA were derived from transposon-encoded TnpB nucleases, most likely, on several independent occasions. Fourth, accessory nucleases in some variants of types I and III toxin and type VI effectors RNases appear to be ultimately derived from toxin nucleases of microbial toxin-antitoxin modules. Fifth, the opposite direction of evolution is manifested in the recruitment of CRISPR-Cas systems by a distinct family of Tn7-like transposons that probably exploit the capacity of CRISPR-Cas to recognize unique DNA sites to facilitate transposition as well as by bacteriophages that employ them to cope with host defense. Additionally, individual Cas proteins, such as the Cas4 nuclease, were recruited by bacteriophages and transposons. The two-sided evolutionary connection between CRISPR-Cas and MGE fits the "guns for hire" paradigm whereby homologous enzymatic machineries, in particular nucleases, are shuttled between MGE and defense systems and are used alternately as means of offense or defense. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2017. This work is written by US Government employees and is in the public

  6. Mobile Genetic Elements and Evolution of CRISPR-Cas Systems: All the Way There and Back

    PubMed Central

    Makarova, Kira S.

    2017-01-01

    Abstract The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) systems of bacterial and archaeal adaptive immunity show multifaceted evolutionary relationships with at least five classes of mobile genetic elements (MGE). First, the adaptation module of CRISPR-Cas that is responsible for the formation of the immune memory apparently evolved from a Casposon, a self-synthesizing transposon that employs the Cas1 protein as the integrase and might have brought additional cas genes to the emerging immunity loci. Second, a large subset of type III CRISPR-Cas systems recruited a reverse transcriptase from a Group II intron, providing for spacer acquisition from RNA. Third, effector nucleases of Class 2 CRISPR-Cas systems that are responsible for the recognition and cleavage of the target DNA were derived from transposon-encoded TnpB nucleases, most likely, on several independent occasions. Fourth, accessory nucleases in some variants of types I and III toxin and type VI effectors RNases appear to be ultimately derived from toxin nucleases of microbial toxin–antitoxin modules. Fifth, the opposite direction of evolution is manifested in the recruitment of CRISPR-Cas systems by a distinct family of Tn7-like transposons that probably exploit the capacity of CRISPR-Cas to recognize unique DNA sites to facilitate transposition as well as by bacteriophages that employ them to cope with host defense. Additionally, individual Cas proteins, such as the Cas4 nuclease, were recruited by bacteriophages and transposons. The two-sided evolutionary connection between CRISPR-Cas and MGE fits the “guns for hire” paradigm whereby homologous enzymatic machineries, in particular nucleases, are shuttled between MGE and defense systems and are used alternately as means of offense or defense. PMID:28985291

  7. An Implementation of the Action Space Concept in Behavioral Analysis.

    ERIC Educational Resources Information Center

    Higgs, Gary K.

    The Contact Action Space (CAS) of an individual, or group of individuals, has a significant impact on the location of activities and the organization of the use of space. Beginning with the most basic components of a CAS, the individual behavior pattern element is developed, and operational variations affecting alignment and configuration are…

  8. Naturally Occurring Off-Switches for CRISPR-Cas9.

    PubMed

    Pawluk, April; Amrani, Nadia; Zhang, Yan; Garcia, Bianca; Hidalgo-Reyes, Yurima; Lee, Jooyoung; Edraki, Alireza; Shah, Megha; Sontheimer, Erik J; Maxwell, Karen L; Davidson, Alan R

    2016-12-15

    CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These "anti-CRISPRs" were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable "off-switches" for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. VIDEO ABSTRACT. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Somatic cell nuclear transfer followed by CRIPSR/CAS9 microinjection results in highly efficient genome editing in cloned pigs

    USDA-ARS?s Scientific Manuscript database

    The domestic pig is an ideal “dual purpose” animal model for agricultural and biomedical research. With the availability of genome editing tools [e.g. clustered regularly interspersed short palindromic repeat (CRISPR) and associated nuclease Cas9 (CRISPR/Cas9)] it is now possible to perform site-sp...

  10. Corrective Action Decision Document/Closure Report for Corrective Action Unit 252: Area 25 Engine Test Stand 1 Decontamination Pad, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    DOE /NV

    This Corrective Action Decision Document/Closure Report (CADD/CR) has been prepared for Corrective Action Unit (CAU) 252: Area 25 Engine Test Stand-1 Decontamination Pad, in accordance with the Federal Facility Agreement and Consent Order (FFACO). Located at the Nevada Test Site in Nevada, CAU 252 consists of only one Corrective Action Site (25-07-04, Decontamination Pad). This CADD/CR identifies and rationalizes the U.S. Department of Energy, Nevada Operations Office's (DOE/NV's) recommendation that no corrective action is deemed necessary at CAU 252. The Corrective Action Decision Document and Closure Report have been combined into one report because the potential contaminants of concern weremore » either not detected during the corrective action investigation or were only present at naturally occurring concentrations. Based on the field results, neither corrective action or a corrective action plan is required at this site. A Notice of Completion to DOE/NV is being requested from the Nevada Division of Environmental Protection for closure of CAU 252, as well as a request that this site be moved from Appendix III to Appendix IV of the FFACO. Further, no use restrictions are required to be placed on this CAU.« less

  11. Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells.

    PubMed

    Liao, Hsin-Kai; Gu, Ying; Diaz, Arturo; Marlett, John; Takahashi, Yuta; Li, Mo; Suzuki, Keiichiro; Xu, Ruo; Hishida, Tomoaki; Chang, Chan-Jung; Esteban, Concepcion Rodriguez; Young, John; Izpisua Belmonte, Juan Carlos

    2015-03-10

    To combat hostile viruses, bacteria and archaea have evolved a unique antiviral defense system composed of clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated genes (Cas). The CRISPR/Cas9 system develops an adaptive immune resistance to foreign plasmids and viruses by creating site-specific DNA double-stranded breaks (DSBs). Here we adapt the CRISPR/Cas9 system to human cells for intracellular defense against foreign DNA and viruses. Using HIV-1 infection as a model, our results demonstrate that the CRISPR/Cas9 system disrupts latently integrated viral genome and provides long-term adaptive defense against new viral infection, expression and replication in human cells. We show that engineered human-induced pluripotent stem cells stably expressing HIV-targeted CRISPR/Cas9 can be efficiently differentiated into HIV reservoir cell types and maintain their resistance to HIV-1 challenge. These results unveil the potential of the CRISPR/Cas9 system as a new therapeutic strategy against viral infections.

  12. CRISPR/Cas9 gene drives in genetically variable and nonrandomly mating wild populations

    PubMed Central

    Drury, Douglas W.; Dapper, Amy L.; Siniard, Dylan J.; Zentner, Gabriel E.; Wade, Michael J.

    2017-01-01

    Synthetic gene drives based on CRISPR/Cas9 have the potential to control, alter, or suppress populations of crop pests and disease vectors, but it is unclear how they will function in wild populations. Using genetic data from four populations of the flour beetle Tribolium castaneum, we show that most populations harbor genetic variants in Cas9 target sites, some of which would render them immune to drive (ITD). We show that even a rare ITD allele can reduce or eliminate the efficacy of a CRISPR/Cas9-based synthetic gene drive. This effect is equivalent to and accentuated by mild inbreeding, which is a characteristic of many disease-vectoring arthropods. We conclude that designing such drives will require characterization of genetic variability and the mating system within and among targeted populations. PMID:28560324

  13. CRISPR/Cas9 gene drives in genetically variable and nonrandomly mating wild populations.

    PubMed

    Drury, Douglas W; Dapper, Amy L; Siniard, Dylan J; Zentner, Gabriel E; Wade, Michael J

    2017-05-01

    Synthetic gene drives based on CRISPR/Cas9 have the potential to control, alter, or suppress populations of crop pests and disease vectors, but it is unclear how they will function in wild populations. Using genetic data from four populations of the flour beetle Tribolium castaneum , we show that most populations harbor genetic variants in Cas9 target sites, some of which would render them immune to drive (ITD). We show that even a rare ITD allele can reduce or eliminate the efficacy of a CRISPR/Cas9-based synthetic gene drive. This effect is equivalent to and accentuated by mild inbreeding, which is a characteristic of many disease-vectoring arthropods. We conclude that designing such drives will require characterization of genetic variability and the mating system within and among targeted populations.

  14. Corrective Action Decision Document/Corrective Action Plan for Corrective Action Unit 97: Yucca Flat/Climax Mine Nevada National Security Site, Nevada, Revision 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Farnham, Irene

    This corrective action decision document (CADD)/corrective action plan (CAP) has been prepared for Corrective Action Unit (CAU) 97, Yucca Flat/Climax Mine, Nevada National Security Site (NNSS), Nevada. The Yucca Flat/Climax Mine CAU is located in the northeastern portion of the NNSS and comprises 720 corrective action sites. A total of 747 underground nuclear detonations took place within this CAU between 1957 and 1992 and resulted in the release of radionuclides (RNs) in the subsurface in the vicinity of the test cavities. The CADD portion describes the Yucca Flat/Climax Mine CAU data-collection and modeling activities completed during the corrective action investigationmore » (CAI) stage, presents the corrective action objectives, and describes the actions recommended to meet the objectives. The CAP portion describes the corrective action implementation plan. The CAP presents CAU regulatory boundary objectives and initial use-restriction boundaries identified and negotiated by DOE and the Nevada Division of Environmental Protection (NDEP). The CAP also presents the model evaluation process designed to build confidence that the groundwater flow and contaminant transport modeling results can be used for the regulatory decisions required for CAU closure. The UGTA strategy assumes that active remediation of subsurface RN contamination is not feasible with current technology. As a result, the corrective action is based on a combination of characterization and modeling studies, monitoring, and institutional controls. The strategy is implemented through a four-stage approach that comprises the following: (1) corrective action investigation plan (CAIP), (2) CAI, (3) CADD/CAP, and (4) closure report (CR) stages.« less

  15. RNA targeting with CRISPR-Cas13.

    PubMed

    Abudayyeh, Omar O; Gootenberg, Jonathan S; Essletzbichler, Patrick; Han, Shuo; Joung, Julia; Belanto, Joseph J; Verdine, Vanessa; Cox, David B T; Kellner, Max J; Regev, Aviv; Lander, Eric S; Voytas, Daniel F; Ting, Alice Y; Zhang, Feng

    2017-10-12

    RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.

  16. New CRISPR–Cas systems from uncultivated microbes

    DOE PAGES

    Burstein, David; Harrington, Lucas B.; Strutt, Steven C.; ...

    2016-12-22

    We present that CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNAmore » extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Lastly, interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.« less

  17. New CRISPR–Cas systems from uncultivated microbes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Burstein, David; Harrington, Lucas B.; Strutt, Steven C.

    We present that CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNAmore » extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Lastly, interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.« less

  18. Efficient CRISPR/Cas9-mediated Targeted Mutagenesis in Populus in the First Generation

    PubMed Central

    Fan, Di; Liu, Tingting; Li, Chaofeng; Jiao, Bo; Li, Shuang; Hou, Yishu; Luo, Keming

    2015-01-01

    Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been applied to edit the plant genome in several herbaceous plant species. However, it remains unknown whether this system can be used for genome editing in woody plants. In this study, we describe the genome editing and targeted gene mutation in a woody species, Populus tomentosa Carr. via the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed to target with distinct poplar genomic sites of the phytoene desaturase gene 8 (PtoPDS) which are followed by the protospacer-adjacent motif (PAM). After Agrobacterium-mediated transformation, obvious albino phenotype was observed in transgenic poplar plants. By analyzing the RNA-guided genome-editing events, 30 out of 59 PCR clones were homozygous mutants, 2 out of 59 were heterozygous mutants and the mutation efficiency at these target sites was estimated to be 51.7%. Our data demonstrate that the Cas9/sgRNA system can be exploited to precisely edit genomic sequence and effectively create knockout mutations in woody plants. PMID:26193631

  19. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9

    PubMed Central

    Mandal, Pankaj K.; Ferreira, Leonardo M. R.; Collins, Ryan; Meissner, Torsten B.; Boutwell, Christian L.; Friesen, Max; Vrbanac, Vladimir; Garrison, Brian S.; Stortchevoi, Alexei; Bryder, David; Musunuru, Kiran; Brand, Harrison; Tager, Andrew M.; Allen, Todd M.; Talkowski, Michael E.; Rossi, Derrick J.; Cowan, Chad A.

    2014-01-01

    SUMMARY Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9 mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multi-lineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy. PMID:25517468

  20. Not all predicted CRISPR-Cas systems are equal: isolated cas genes and classes of CRISPR like elements.

    PubMed

    Zhang, Quan; Ye, Yuzhen

    2017-02-06

    The CRISPR-Cas systems in prokaryotes are RNA-guided immune systems that target and deactivate foreign nucleic acids. A typical CRISPR-Cas system consists of a CRISPR array of repeat and spacer units, and a locus of cas genes. The CRISPR and the cas locus are often located next to each other in the genomes. However, there is no quantitative estimate of the co-location. In addition, ad-hoc studies have shown that some non-CRISPR genomic elements contain repeat-spacer-like structures and are mistaken as CRISPRs. Using available genome sequences, we observed that a significant number of genomes have isolated cas loci and/or CRISPRs. We found that 11%, 22% and 28% of the type I, II and III cas loci are isolated (without CRISPRs in the same genomes at all or with CRISPRs distant in the genomes), respectively. We identified a large number of genomic elements that superficially reassemble CRISPRs but don't contain diverse spacers and have no companion cas genes. We called these elements false-CRISPRs and further classified them into groups, including tandem repeats and Staphylococcus aureus repeat (STAR)-like elements. This is the first systematic study to collect and characterize false-CRISPR elements. We demonstrated that false-CRISPRs could be used to reduce the false annotation of CRISPRs, therefore showing them to be useful for improving the annotation of CRISPR-Cas systems.

  1. A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.

    PubMed

    Ma, Xingliang; Zhang, Qunyu; Zhu, Qinlong; Liu, Wei; Chen, Yan; Qiu, Rong; Wang, Bin; Yang, Zhongfang; Li, Heying; Lin, Yuru; Xie, Yongyao; Shen, Rongxin; Chen, Shuifu; Wang, Zhi; Chen, Yuanling; Guo, Jingxin; Chen, Letian; Zhao, Xiucai; Dong, Zhicheng; Liu, Yao-Guang

    2015-08-01

    CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  2. CRISPR-Cas9 vectors for genome editing and host engineering in the baculovirus-insect cell system.

    PubMed

    Mabashi-Asazuma, Hideaki; Jarvis, Donald L

    2017-08-22

    The baculovirus-insect cell system (BICS) has been widely used to produce many different recombinant proteins for basic research and is being used to produce several biologics approved for use in human or veterinary medicine. Early BICS were technically complex and constrained by the relatively primordial nature of insect cell protein glycosylation pathways. Since then, recombination has been used to modify baculovirus vectors-which has simplified the system-and transform insect cells, which has enhanced its protein glycosylation capabilities. Now, CRISPR-Cas9 tools for site-specific genome editing are needed to facilitate further improvements in the BICS. Thus, in this study, we used various insect U6 promoters to construct CRISPR-Cas9 vectors and assessed their utility for site-specific genome editing in two insect cell lines commonly used as hosts in the BICS. We demonstrate the use of CRISPR-Cas9 to edit an endogenous insect cell gene and alter protein glycosylation in the BICS.

  3. Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.

    PubMed

    Rauch, Benjamin J; Silvis, Melanie R; Hultquist, Judd F; Waters, Christopher S; McGregor, Michael J; Krogan, Nevan J; Bondy-Denomy, Joseph

    2017-01-12

    Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific "anti-CRISPRs" present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. [Construction of EZH2 Knockout Animal Model by CRISPR/Cas9 Technology].

    PubMed

    Meng, Fanrong; Zhao, Dan; Zhou, Qinghua; Liu, Zhe

    2018-05-20

    It has been proven that CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9) system was the modern gene-editing technology through the constitutive expression of nucleases Cas9 in the mammalian, which binds to the specific site in the genome mediated by single-guide RNA (sgRNA) at desired genomic loci. The aim of this study is that the animal model of EZH2 gene knockout was constructed using CRISPR/Cas9 technology. In this study, we designed two single-guide RNAs targeting the Exon3 and Exon4 of EZH2 gene. Then, their gene-targeting efficiency were detected by SURVEYOR assay. The lentivirus was perfused into the lungs of mice by using a bronchial tube and detected by immunohistochemistry and qRT-PCR. The experimental results of NIH-3T3 cells verify that the designed sgEZH2 can efficiently effect the cleavage of target DNA by Cas9 in vitro. The immunohistochemistry and qRT-PCR results showed that the EZH2 expression in experimental group was significantly decreased in the mouse lung tissue. The study successfully designed two sgRNA which can play a knock-out EZH2 function. An EZH2 knockout animal model was successfully constructed by CRISPR/Cas9 system, and it will be an effective animal model for studying the functions and mechanisms of EZH2.

  5. Assembly and Multiplex Genome Integration of Metabolic Pathways in Yeast Using CasEMBLR.

    PubMed

    Jakočiūnas, Tadas; Jensen, Emil D; Jensen, Michael K; Keasling, Jay D

    2018-01-01

    Genome integration is a vital step for implementing large biochemical pathways to build a stable microbial cell factory. Although traditional strain construction strategies are well established for the model organism Saccharomyces cerevisiae, recent advances in CRISPR/Cas9-mediated genome engineering allow much higher throughput and robustness in terms of strain construction. In this chapter, we describe CasEMBLR, a highly efficient and marker-free genome engineering method for one-step integration of in vivo assembled expression cassettes in multiple genomic sites simultaneously. CasEMBLR capitalizes on the CRISPR/Cas9 technology to generate double-strand breaks in genomic loci, thus prompting native homologous recombination (HR) machinery to integrate exogenously derived homology templates. As proof-of-principle for microbial cell factory development, CasEMBLR was used for one-step assembly and marker-free integration of the carotenoid pathway from 15 exogenously supplied DNA parts into three targeted genomic loci. As a second proof-of-principle, a total of ten DNA parts were assembled and integrated in two genomic loci to construct a tyrosine production strain, and at the same time knocking out two genes. This new method complements and improves the field of genome engineering in S. cerevisiae by providing a more flexible platform for rapid and precise strain building.

  6. Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering.

    PubMed

    Ui-Tei, Kumiko; Maruyama, Shohei; Nakano, Yuko

    2017-06-01

    Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

  7. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

    PubMed

    Gagnon, James A; Valen, Eivind; Thyme, Summer B; Huang, Peng; Akhmetova, Laila; Ahkmetova, Laila; Pauli, Andrea; Montague, Tessa G; Zimmerman, Steven; Richter, Constance; Schier, Alexander F

    2014-01-01

    The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.

  8. Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.

    PubMed

    Rivera-Torres, Natalia; Banas, Kelly; Bialk, Pawel; Bloh, Kevin M; Kmiec, Eric B

    2017-01-01

    CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and

  9. Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides

    PubMed Central

    Rivera-Torres, Natalia; Bialk, Pawel; Bloh, Kevin M.; Kmiec, Eric B.

    2017-01-01

    CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and

  10. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas.

    PubMed

    Polte, T R; Hanks, S K

    1995-11-07

    The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, we used a two-hybrid screen to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), p130Cas, and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of p130Cas. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse p130Cas amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that p130Cas and FAK are associated in mouse fibroblasts. The stable interaction between p130Cas and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Src homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen.

  11. DOUBLE TRACKS Test Site interim corrective action plan

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    The DOUBLE TRACKS site is located on Range 71 north of the Nellis Air Force Range, northwest of the Nevada Test Site (NTS). DOUBLE TRACKS was the first of four experiments that constituted Operation ROLLER COASTER. On May 15, 1963, weapons-grade plutonium and depleted uranium were dispersed using 54 kilograms of trinitrotoluene (TNT) explosive. The explosion occurred in the open, 0.3 m above the steel plate. No fission yield was detected from the test, and the total amount of plutonium deposited on the ground surface was estimated to be between 980 and 1,600 grams. The test device was composed primarilymore » of uranium-238 and plutonium-239. The mass ratio of uranium to plutonium was 4.35. The objective of the corrective action is to reduce the potential risk to human health and the environment and to demonstrate technically viable and cost-effective excavation, transportation, and disposal. To achieve these objectives, Bechtel Nevada (BN) will remove soil with a total transuranic activity greater then 200 pCI/g, containerize the soil in ``supersacks,`` transport the filled ``supersacks`` to the NTS, and dispose of them in the Area 3 Radioactive Waste Management Site. During this interim corrective action, BN will also conduct a limited demonstration of an alternative method for excavation of radioactive near-surface soil contamination.« less

  12. CAS-Induced Difficulties in Learning Mathematics?

    ERIC Educational Resources Information Center

    Jankvist, Uffe Thomas; Misfeldt, Morten

    2015-01-01

    In recent years computer algebra systems (CAS) have become an integrated part of the upper secondary school mathematics program. Despite the many positive possibilities of CAS, there also seems to be a flip side of the coin in relation to actual difficulties in learning mathematics, not least because a strong dependence on CAS for mathematical…

  13. [CAS General Standards 2012

    ERIC Educational Resources Information Center

    Council for the Advancement of Standards in Higher Education, 2011

    2011-01-01

    The mission of the Council for the Advancement of Standards in Higher Education (CAS) is to promote the improvement of programs and services to enhance the quality of student learning and development. CAS is a consortium of professional associations who work collaboratively to develop and promulgate standards and guidelines and to encourage…

  14. Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells.

    PubMed

    Sternburg, Erin L; Dias, Kristen C; Karginov, Fedor V

    2017-06-16

    The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

  15. Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus.

    PubMed

    Merkle, Florian T; Neuhausser, Werner M; Santos, David; Valen, Eivind; Gagnon, James A; Maas, Kristi; Sandoe, Jackson; Schier, Alexander F; Eggan, Kevin

    2015-05-12

    The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  16. The Central Nervous System Sites Mediating the Orexigenic Actions of Ghrelin

    PubMed Central

    Mason, B.L.; Wang, Q.; Zigman, J.M.

    2014-01-01

    The peptide hormone ghrelin is important for both homeostatic and hedonic eating behaviors, and its orexigenic actions occur mainly via binding to the only known ghrelin receptor, the growth hormone secretagogue receptor (GHSR). GHSRs are located in several distinct regions of the central nervous system. This review discusses those central nervous system sites that have been found to play critical roles in the orexigenic actions of ghrelin, including hypothalamic nuclei, the hippocampus, the amygdala, the caudal brain stem, and midbrain dopaminergic neurons. Hopefully, this review can be used as a stepping stone for the reader wanting to gain a clearer understanding of the central nervous system sites of direct ghrelin action on feeding behavior, and as inspiration for future studies to provide an even-more-detailed map of the neurocircuitry controlling eating and body weight. PMID:24111557

  17. Using CRISPR-Cas systems as antimicrobials.

    PubMed

    Bikard, David; Barrangou, Rodolphe

    2017-06-01

    Although CRISPR-Cas systems naturally evolved to provide adaptive immunity in bacteria and archaea, Cas nucleases can be co-opted to target chromosomal sequences rather than invasive genetic elements. Although genome editing is the primary outcome of self-targeting using CRISPR-based technologies in eukaryotes, self-targeting by CRISPR is typically lethal in bacteria. Here, we discuss how DNA damage introduced by Cas nucleases in bacteria can efficiently and specifically lead to plasmid curing or drive cell death. Specifically, we discuss how various CRISPR-Cas systems can be engineered and delivered using phages or phagemids as vectors. These principles establish CRISPR-Cas systems as potent and programmable antimicrobials, and open new avenues for the development of CRISPR-based tools for selective removal of bacterial pathogens and precise microbiome composition alteration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Corrective Action Decision Document for Corrective Action Unit 224: Decon Pad and Septic Systems Nevada Test Site, Nevada, Rev. No.: 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    David A. Strand

    2005-05-01

    This Corrective Action Decision Document has been prepared for Corrective Action Unit (CAU) 224, Decon Pad and Septic Systems, in Areas 2, 3, 5, 6, 11, and 23 of the Nevada Test Site, Nevada, in accordance with the ''Federal Facility Agreement and Consent Order'' (1996). Corrective Action Unit 224 is comprised of the following corrective action sites (CASs): (1) 02-04-01, Septic Tank (Buried); (2) 03-05-01, Leachfield; (3) 05-04-01, Septic Tanks (4)/Discharge Area; (4) 06-03-01, Sewage Lagoons (3); (5) 06-05-01, Leachfield; (6) 06-17-04, Decon Pad and Wastewater Catch; (7) 06-23-01, Decon Pad Discharge Piping; (8) 11-04-01, Sewage Lagoon; and (9) 23-05-02,more » Leachfield. The purpose of this Corrective Action Decision Document is to identify and provide the rationale for the recommendation of a corrective action alternative for the nine CASs within CAU 224. Corrective action investigation activities were performed from August 10, 2004, through January 18, 2005, as set forth in the CAU 224 Corrective Action Investigation Plan.« less

  19. No Further Remedial Action Planned Decision Document for Site 3.

    DTIC Science & Technology

    1998-04-01

    INSTALLATION RESTORATION PROGRAM No FURTHER REMEDIAL ACTION PLANNED DECISION DOCUMENT FOR SITE 3 FINAL MICHIGAN AIR NATIONAL GUARD ALPENA ...COMBAT READINESS TRAINING CENTER ALPENA , MICHIGAN April 1998 Air National Guard Andrews AFB, Maryland &nc QUALITY IMSmm«^ 19980519 204 XA REPORT...Document for Site 3 at Alpena CRTC, Alpena , MI. 6. AUTHOR(S) N/A 5. FUNDING NUMBERS 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Montgomery

  20. Evolution and classification of the CRISPR-Cas systems

    PubMed Central

    S. Makarova, Kira; H. Haft, Daniel; Barrangou, Rodolphe; J. J. Brouns, Stan; Charpentier, Emmanuelle; Horvath, Philippe; Moineau, Sylvain; J. M. Mojica, Francisco; I. Wolf, Yuri; Yakunin, Alexander F.; van der Oost, John; V. Koonin, Eugene

    2012-01-01

    The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR–Cas systems and Cas proteins. Three major types of CRISPR–Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR–Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a `polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR–cas loci. PMID:21552286

  1. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein.

    PubMed

    Tang, Lichun; Zeng, Yanting; Du, Hongzi; Gong, Mengmeng; Peng, Jin; Zhang, Buxi; Lei, Ming; Zhao, Fang; Wang, Weihua; Li, Xiaowei; Liu, Jianqiao

    2017-06-01

    Previous works using human tripronuclear zygotes suggested that the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system could be a tool in correcting disease-causing mutations. However, whether this system was applicable in normal human (dual pronuclear, 2PN) zygotes was unclear. Here we demonstrate that CRISPR/Cas9 is also effective as a gene-editing tool in human 2PN zygotes. By injection of Cas9 protein complexed with the appropriate sgRNAs and homology donors into one-cell human embryos, we demonstrated efficient homologous recombination-mediated correction of point mutations in HBB and G6PD. However, our results also reveal limitations of this correction procedure and highlight the need for further research.

  2. CRISPR-Cas: biology, mechanisms and relevance

    PubMed Central

    Hille, Frank

    2016-01-01

    Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes—termed spacers—into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent. This article is part of the themed issue ‘The new bacteriology’. PMID:27672148

  3. CRISPR-Cas: biology, mechanisms and relevance.

    PubMed

    Hille, Frank; Charpentier, Emmanuelle

    2016-11-05

    Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes-termed spacers-into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent.This article is part of the themed issue 'The new bacteriology'. © 2016 The Authors.

  4. Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention.

    PubMed

    Zhang, Qiang; Xing, Hui-Li; Wang, Zhi-Ping; Zhang, Hai-Yan; Yang, Fang; Wang, Xue-Chen; Chen, Qi-Jun

    2018-03-01

    We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations. Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.

  5. Notification: Evaluation of Time-Critical Removal Actions for Superfund Sites

    EPA Pesticide Factsheets

    Project #OPE-FY15-0019, February 4, 2015. The EPA OIG plans to begin preliminary research on the evaluation of the effectiveness of time-critical removal actions for Superfund sites on February 24, 2015.

  6. Corrective Action Investigation Plan for Corrective Action Unit 414: Clean Slate III Plutonium Dispersion (TTR) Tonopah Test Range, Nevada, Revision 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    Corrective Action Unit (CAU) 414 is located on the Tonopah Test Range, which is approximately 130 miles northwest of Las Vegas, Nevada, and approximately 40 miles southeast of Tonopah, Nevada. The CAU 414 site consists of the release of radionuclides to the surface and shallow subsurface from the conduct of the Clean Slate III (CSIII) storage–transportation test conducted on June 9, 1963. CAU 414 includes one corrective action site (CAS), TA-23-03CS (Pu Contaminated Soil). The known releases at CAU 414 are the result of the atmospheric dispersal of contamination from the 1963 CSIII test. The CSIII test was a nonnuclearmore » detonation of a nuclear device located inside a reinforced concrete bunker covered with 8 feet of soil. This test dispersed radionuclides, primarily uranium and plutonium, on the ground surface. The presence and nature of contamination at CAU 414 will be evaluated based on information collected from a corrective action investigation (CAI). The investigation is based on the data quality objectives (DQOs) developed on June 7, 2016, by representatives of the Nevada Division of Environmental Protection; the U.S. Air Force; and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Field Office. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective action alternatives for CAU 414.« less

  7. Engineering Translational Activators with CRISPR-Cas System.

    PubMed

    Du, Pei; Miao, Chensi; Lou, Qiuli; Wang, Zefeng; Lou, Chunbo

    2016-01-15

    RNA parts often serve as critical components in genetic engineering. Here we report a design of translational activators which is composed of an RNA endoribonuclease (Csy4) and two exchangeable RNA modules. Csy4, a member of Cas endoribonuclease, cleaves at a specific recognition site; this cleavage releases a cis-repressive RNA module (crRNA) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. Unlike small RNA as a translational activator, the endoribonuclease-based activator is able to efficiently unfold the perfect RBS-crRNA pairing. As an exchangeable module, the crRNA-RBS duplex was forwardly and reversely engineered to modulate the dynamic range of translational activity. We further showed that Csy4 and its recognition site, together as a module, can also be replaced by orthogonal endoribonuclease-recognition site homologues. These modularly structured, high-performance translational activators would endow the programming of gene expression in the translation level with higher feasibility.

  8. Rational Design of Mini-Cas9 for Transcriptional Activation.

    PubMed

    Ma, Dacheng; Peng, Shuguang; Huang, Weiren; Cai, Zhiming; Xie, Zhen

    2018-04-20

    Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.

  9. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false CAS applicability. 30.201-1 Section 30.201-1 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS...

  10. Repurposing the CRISPR-Cas9 system for targeted DNA methylation.

    PubMed

    Vojta, Aleksandar; Dobrinić, Paula; Tadić, Vanja; Bočkor, Luka; Korać, Petra; Julg, Boris; Klasić, Marija; Zoldoš, Vlatka

    2016-07-08

    Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co-expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Addendum to the Closure Report for Corrective Action Unit 427: Area 3 Septic Waste Systems 2, 6, Tonopah Test Range, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the April 1999, Closure Report for Corrective Action Unit 427: Area 3 Septic Waste Systems 2, 6, Tonopah Test Range, Nevada as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. The approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modificationmore » document, this addendum consists of: • This cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the cancellation of the URs for: • CAS 03-05-002-SW02, Septic Waste System • CAS 03-05-002-SW06, Septic Waste System These URs were established as part of Federal Facility Agreement and Consent Order (FFACO) corrective actions and were based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since these URs were established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, these URs were re-evaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006c). This re-evaluation consisted of comparing the original data (used to define the need for the URs) to risk-based final action levels (FALs) developed using the current Industrial Sites RBCA process. The re

  12. A novel sgRNA selection system for CRISPR-Cas9 in mammalian cells.

    PubMed

    Zhang, Haiwei; Zhang, Xixi; Fan, Cunxian; Xie, Qun; Xu, Chengxian; Zhao, Qun; Liu, Yongbo; Wu, Xiaoxia; Zhang, Haibing

    2016-03-18

    CRISPR-Cas9 mediated genome editing system has been developed as a powerful tool for elucidating the function of genes through genetic engineering in multiple cells and organisms. This system takes advantage of a single guide RNA (sgRNA) to direct the Cas9 endonuclease to a specific DNA site to generate mutant alleles. Since the targeting efficiency of sgRNAs to distinct DNA loci can vary widely, there remains a need for a rapid, simple and efficient sgRNA selection method to overcome this limitation of the CRISPR-Cas9 system. Here we report a novel system to select sgRNA with high efficacy for DNA sequence modification by a luciferase assay. Using this sgRNAs selection system, we further demonstrated successful examples of one sgRNA for generating one gene knockout cell lines where the targeted genes are shown to be functionally defective. This system provides a potential application to optimize the sgRNAs in different species and to generate a powerful CRISPR-Cas9 genome-wide screening system with minimum amounts of sgRNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. RNA Editing with CRISPR-Cas13

    PubMed Central

    Cox, David B.T.; Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Franklin, Brian; Kellner, Max J.; Joung, Julia; Zhang, Feng

    2017-01-01

    Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided RNases Cas13. Here, we profile Type VI systems to engineer a Cas13 ortholog capable of robust knockdown and demonstrate RNA editing by using catalytically-inactive Cas13 (dCas13) to direct adenosine to inosine deaminase activity by ADAR2 to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineer this system to create a high specificity variant, REPAIRv2, that is 919 times more specific than REPAIRv1 as well as minimize the system to ease viral delivery. REPAIR presents a promising RNA editing platform with broad applicability for research, therapeutics, and biotechnology. PMID:29070703

  14. The Reverse Transcriptases Associated with CRISPR-Cas Systems.

    PubMed

    Toro, Nicolás; Martínez-Abarca, Francisco; González-Delgado, Alejandro

    2017-08-02

    CRISPR (clustered regularly interspaced short palindromic repeats) and associated proteins (Cas) act as adaptive immune systems in bacteria and archaea. Some CRISPR-Cas systems have been found to be associated with putative reverse transcriptases (RT), and an RT-Cas1 fusion associated with a type III-B system has been shown to acquire RNA spacers in vivo. Nevertheless, the origin and evolutionary relationships of these RTs and associated CRISPR-Cas systems remain largely unknown. We performed a comprehensive phylogenetic analysis of these RTs and associated Cas1 proteins, and classified their CRISPR-Cas modules. These systems were found predominantly in bacteria, and their presence in archaea may be due to a horizontal gene transfer event. These RTs cluster into 12 major clades essentially restricted to particular phyla, suggesting host-dependent functioning. The RTs and associated Cas1 proteins may have largely coevolved. They are, therefore, subject to the same selection pressures, which may have led to coadaptation within particular protein complexes. Furthermore, our results indicate that the association of an RT with a CRISPR-Cas system has occurred on multiple occasions during evolution.

  15. Spectroscopic studies of three Cepheids with high positive pulsation period increments: SZ Cas, BY Cas, and RU Sct

    NASA Astrophysics Data System (ADS)

    Usenko, I. A.; Klochkova, V. G.

    2015-07-01

    Three high-resolution spectra have been taken at different times with the 6-m SAO RAS telescope (LYNX and PFES spectrographs) for three Cepheids exhibiting high positive period increments: the small-amplitude (DCEPS) SZ Cas and BY Cas and the classical (DCEP) RU Sct. SZ Cas and RU Sct are members of the Galactic open clusters χ and h Per and Trump 35, respectively. Analysis of the spectra has shown that the interstellar Na I D1 and D2 lines in all objects are considerably stronger than the atmospheric ones and are redshifted in SZ Cas and BY Cas and blushifted in RU Sct. The core of the H α absorption line in BY Cas has an asymmetric knifelike shape, while RU Sct exhibits an intense emission in the blue wing of this line. Such phenomena are observed in long-period Cepheids and bright hypergiants with an extended envelope. In this case, the strong Mg Ib 5183.62 Å and Ba II 5853.67, 6141.713, and 6496.90 Å lines with low χlow in SZ Cas and RU Sct also show characteristic knifelike profiles with an asymmetry in the red region, while the Ba II 4934.095 Å line shows similar profiles in the blue one. The absorption lines of neutral atoms and singly ionized metals with different lowerlevel excitation potentials exhibit different degrees of asymmetry: from a pronounced one with secondary components in BY Cas (similar to those in the small-amplitude Cepheid BG Cru pulsating in the first overtone and having an envelope) to its insignificance or virtual absence in SZ Cas and RU Sct. Analysis of the secular changes in mean T eff determined from photometric color indices and spectra over the last 55 years for these stars has revealed periodic fluctuations of 200 K for SZ Cas and BY Cas and 500 K for RU Sct. For SZ Cas and RU Sct, T eff determined in some years from some color indices show much lower values, which together with the temperature fluctuations can be associated with mass loss and dust formation. Based on these facts, we hypothesize the existence of

  16. Closure Report for Corrective Action Unit 426: Cactus Spring Waste Trenches, Tonopah Test Range, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dave Madsen

    This Closure Report provides the documentation for closure of the Cactus Spring Waste Trenches Corrective Action Unit (CAU) 426. The site is located on the Tonopah Test Range, approximately 225 kilometers northwest of Las Vegas, NV. CAU 426 consists of one corrective action site (CAS) which is comprised of four waste trenches. The trenches were excavated to receive solid waste generated in support of Operation Roller Coaster, primary the Double Tracks Test in 1963, and were subsequently backfilled. The Double Tracks Test involved use of live animals to assess the biological hazards associated with the nonnuclear detonation of plutonium-bearing devices.more » The Nevada Division of Environmental Protection approved Corrective Action Plan (CAP)which proposed ''capping'' methodology. The closure activities were completed in accordance with the approved CAP and consisted of constructing an engineered cover in the area of the trenches, constructing/planting a vegetative cover, installing a perimeter fence and signs, implementing restrictions on future use, and preparing a Post-Closure Monitoring Plan.« less

  17. Maintenance Plan for the Composite Analysis of the Hanford Site, Southeast Washington.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lehman, L. L.; Nichols, W. E.

    The U.S. Department of Energy (DOE) manuals for radioactive waste management, DOE M 435.1-1 Chg 21 and DOE-STD-5002-2017, require that the Hanford Site maintain site performance assessments and composite analyses (CAs). This document describes the plan for maintaining the CA that supports waste disposal and remedial actions for the Hanford Site. An initial CA of the site was issued in 1998, conditionally approved in 1999, received further analysis to satisfy conditions in an addendum in 2001, and was approved in 2002. This document meets the maintenance plan requirements described in DOE M 435.1-1 Chg 2 and DOE-STD-5002-2017 and implements themore » requirements of the disposal authorization related to the CA for the U.S. Department of Energy, Richland Operations Office, the responsible field office, and its contractors.« less

  18. Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation.

    PubMed

    Kieper, Sebastian N; Almendros, Cristóbal; Behler, Juliane; McKenzie, Rebecca E; Nobrega, Franklin L; Haagsma, Anna C; Vink, Jochem N A; Hess, Wolfgang R; Brouns, Stan J J

    2018-03-27

    CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and observed that cas4 is strictly required for the selection of targets with protospacer adjacent motifs (PAMs) conferring I-D CRISPR interference in the native host Synechocystis. We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM. Introducing functional spacers that target DNA sequences with the correct PAM is key to successful CRISPR interference, providing a better chance of surviving infection by mobile genetic elements. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Disabling Cas9 by an anti-CRISPR DNA mimic.

    PubMed

    Shin, Jiyung; Jiang, Fuguo; Liu, Jun-Jie; Bray, Nicolas L; Rauch, Benjamin J; Baik, Seung Hyun; Nogales, Eva; Bondy-Denomy, Joseph; Corn, Jacob E; Doudna, Jennifer A

    2017-07-01

    CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

  20. Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System

    PubMed Central

    Cooper, Lauren A.; Stringer, Anne M.

    2018-01-01

    ABSTRACT In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5′ end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing. PMID:29666291

  1. Transformation of OODT CAS to Perform Larger Tasks

    NASA Technical Reports Server (NTRS)

    Mattmann, Chris; Freeborn, Dana; Crichton, Daniel; Hughes, John; Ramirez, Paul; Hardman, Sean; Woollard, David; Kelly, Sean

    2008-01-01

    A computer program denoted OODT CAS has been transformed to enable performance of larger tasks that involve greatly increased data volumes and increasingly intensive processing of data on heterogeneous, geographically dispersed computers. Prior to the transformation, OODT CAS (also alternatively denoted, simply, 'CAS') [wherein 'OODT' signifies 'Object-Oriented Data Technology' and 'CAS' signifies 'Catalog and Archive Service'] was a proven software component used to manage scientific data from spaceflight missions. In the transformation, CAS was split into two separate components representing its canonical capabilities: file management and workflow management. In addition, CAS was augmented by addition of a resource-management component. This third component enables CAS to manage heterogeneous computing by use of diverse resources, including high-performance clusters of computers, commodity computing hardware, and grid computing infrastructures. CAS is now more easily maintainable, evolvable, and reusable. These components can be used separately or, taking advantage of synergies, can be used together. Other elements of the transformation included addition of a separate Web presentation layer that supports distribution of data products via Really Simple Syndication (RSS) feeds, and provision for full Resource Description Framework (RDF) exports of metadata.

  2. Chronic arsenic intoxication diagnostic score (CAsIDS).

    PubMed

    Dani, Sergio Ulhoa; Walter, Gerhard Franz

    2018-01-01

    Arsenic and its compounds are well-established, potent, environmentally widespread and persistent toxicants with metabolic, genotoxic, mutagenic, teratogenic, epigenetic and carcinogenic effects. Arsenic occurs naturally in the Earth's crust, but anthropogenic arsenic emissions have surmounted the emissions from important natural sources such as volcanism. Inorganic arsenicals exhibit acute and chronic toxicities in virtually all cell types and tissues, and hence arsenic intoxication affects multiple systems. Whereas acute arsenic intoxication is rare and relatively easy to diagnose, chronic arsenic intoxication (CAsI) is common but goes often misdiagnosed. Based on a review of the literature as well as our own clinical experience, we propose a chronic arsenic intoxication diagnostic score (CAsIDS). A distinctive feature of CAsIDS is the use of bone arsenic load as an essential criterion for the individual risk assessment of chronic arsenic intoxication, combined with a systemic clinical assessment. We present clinical examples where CAsIDS is applied for the diagnosis of CAsI, review the main topics of the toxicity of arsenic in different cell and organ systems and discuss the therapy and prevention of disease caused or aggravated by chronic arsenic intoxication. CAsIDS can help physicians establish the diagnosis of CAsI and associated conditions. Copyright © 2017 John Wiley & Sons, Ltd.

  3. Sulfonamide inhibition studies of two β-carbonic anhydrases from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2.

    PubMed

    Vullo, Daniela; Lehneck, Ronny; Pöggeler, Stefanie; Supuran, Claudiu T

    2018-12-01

    The two β-carbonic anhydrases (CAs, EC 4.2.1.1) recently cloned and purified from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2, were investigated for their inhibition with a panel of 39 aromatic, heterocyclic, and aliphatic sulfonamides and one sulfamate, many of which are clinically used agents. CAS1 was efficiently inhibited by tosylamide, 3-fluorosulfanilamide, and 3-chlorosulfanilamide (K I s in the range of 43.2-79.6 nM), whereas acetazolamide, methazolamide, topiramate, ethoxzolamide, dorzolamide, and brinzolamide were medium potency inhibitors (K I s in the range of 360-445 nM). CAS2 was less sensitive to sulfonamide inhibitors. The best CAS2 inhibitors were 5-amino-1,3,4-thiadiazole-2-sulfonamide (the deacetylated acetazolamide precursor) and 4-hydroxymethyl-benzenesulfonamide, with K I s in the range of 48.1-92.5 nM. Acetazolamide, dorzolamide, ethoxzolamide, topiramate, sulpiride, indisulam, celecoxib, and sulthiame were medium potency CAS2 inhibitors (K I s of 143-857 nM). Many other sulfonamides showed affinities in the high micromolar range or were ineffective as CAS1/2 inhibitors. Small changes in the structure of the inhibitor led to important differences of the activity. As these enzymes may show applications for the removal of anthropically generated polluting gases, finding modulators of their activity may be crucial for designing environmental-friendly CO 2 capture processes.

  4. CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells.

    PubMed

    Bishop, Kathleen A; Harrington, Anne; Kouranova, Evguenia; Weinstein, Edward J; Rosen, Clifford J; Cui, Xiaoxia; Liaw, Lucy

    2016-07-07

    Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation. Copyright © 2016 Bishop et al.

  5. The Power of CRISPR-Cas9-Induced Genome Editing to Speed Up Plant Breeding

    PubMed Central

    Wang, Wenqin; Le, Hien T. T.

    2016-01-01

    Genome editing with engineered nucleases enabling site-directed sequence modifications bears a great potential for advanced plant breeding and crop protection. Remarkably, the RNA-guided endonuclease technology (RGEN) based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) is an extremely powerful and easy tool that revolutionizes both basic research and plant breeding. Here, we review the major technical advances and recent applications of the CRISPR-Cas9 system for manipulation of model and crop plant genomes. We also discuss the future prospects of this technology in molecular plant breeding. PMID:28097123

  6. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kennedy, Edward M.; Cullen, Bryan R., E-mail: bryan.cullen@duke.edu

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called singlemore » guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA

  7. RNA editing with CRISPR-Cas13.

    PubMed

    Cox, David B T; Gootenberg, Jonathan S; Abudayyeh, Omar O; Franklin, Brian; Kellner, Max J; Joung, Julia; Zhang, Feng

    2017-11-24

    Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology. Copyright © 2017, American Association for the Advancement of Science.

  8. Hot News: Gene Therapy with CRISPR/Cas9 Coming to Age for HIV Cure.

    PubMed

    Soriano, Vicente

    2017-01-01

    The huge success of current antiretroviral therapy is mediated by a triple effect: (i) Halting progression to AIDS in infected persons; (ii) reducing the risk of transmission to contacts (treatment as prevention); and (iii) minimizing the risk of HIV acquisition treating uninfected persons at risk (pre-exposure prophylaxis). However, UNAIDS has estimated that only 70% of infected people globally are diagnosed, only 53% are treated, and overall 44% have undetectable viral load, which is the necessary request for ensuring any antiretroviral benefit. Thus, with 37 million people currently living with HIV worldwide and more than 2 million new infections per year, the prospects for global HIV eradication are far on the horizon. Over the past couple of years, rapid development has been seen for technologies enabling modification of gene expression, either by direct inhibition by RNA interference (RNAi) or by genomic modification at DNA level. In particular, genome-editing endonucleases have significantly improved our ability to make precise changes in the DNA of eukaryotic cells. Notably, firstgeneration genome-editing technologies (i.e., ZFNs and TALENs) have been replaced by clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), which work with a short guide RNA (gRNA) to hybridize to a target DNA site and recruit the Cas9 endonuclease. Once integrated into the host genome, HIV gene expression is regulated by the LTR promoter. Hypothetically, gene editing of the HIV promoter might have the potential to deactivate viral transcription by the introduction of mutations or fragment excision. HIV gene therapy progressed very slowly until recent breakthroughs in gene-editing methods using CRISPR/Cas9 (Liao et al. Nat Commun 2015;6:6413). Using a shorter version of the Cas9 endonuclease ensembled into an adenoviral vector, critical segments of thAQ!e viral DNA genome spanning between the LTR and gag regions were successfully removed in HIV transgenic mice

  9. Effects of Using a Computer Algebra System (CAS) on Junior College Students' Attitudes towards CAS and Achievement in Mathematics

    ERIC Educational Resources Information Center

    Leng, Ng Wee; Choo, Kwee Tiow; Soon, Lau Hock; Yi-Huak, Koh; Sun, Yap Yew

    2005-01-01

    This study examines the effects of using Texas Instruments' Voyage 200 calculator (V200), a graphing calculator with a built-in computer algebra system (CAS), on attitudes towards CAS and achievement in mathematics of junior college students (17 year olds). Students' attitudes towards CAS were examined using a 40-item Likert-type instrument…

  10. Final Corrective Action Study for the Former CCC/USDA Facility in Hanover, Kansas

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    LaFreniere, Lorraine M.

    Low concentrations of carbon tetrachloride in groundwater and vapor intrusion into a limited number of residences (attributable to the contaminant concentrations in groundwater) have been identified in Hanover, Kansas, at and near a grain storage facility formerly leased and operated by the Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA). At the request of the Kansas Department of Health and Environment (KDHE 2009h), the CCC/USDA has prepared this Corrective Action Study (CAS) for the facility. The CAS examines corrective actions to address the contamination in groundwater and soil vapor.

  11. [CRISPR/CAS9, the King of Genome Editing Tools].

    PubMed

    Bannikov, A V; Lavrov, A V

    2017-01-01

    The discovery of CRISPR/Cas9 brought a hope for having an efficient, reliable, and readily available tool for genome editing. CRISPR/Cas9 is certainly easy to use, while its efficiency and reliability remain the focus of studies. The review describes the general principles of the organization and function of Cas nucleases and a number of important issues to be considered while planning genome editing experiments with CRISPR/Cas9. The issues include evaluation of the efficiency and specificity for Cas9, sgRNA selection, Cas9 variants designed artificially, and use of homologous recombination and nonhomologous end joining in DNA editing.

  12. Nuclear facility decommissioning and site remedial actions: A selected bibliography, Vol. 18. Part 2. Indexes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    1997-09-01

    This bibliography contains 3638 citations with abstracts of documents relevant to environmental restoration, nuclear facility decontamination and decommissioning (D&D), uranium mill tailings management, and site remedial actions. This report is the eighteenth in a series of bibliographies prepared annually for the U.S. Department of Energy (DOE) Office of Environmental Restoration. Citations to foreign and domestic literature of all types - technical reports, progress reports, journal articles, symposia proceedings, theses, books, patents, legislation, and research project descriptions - have been included in Part 1 of the report. The bibliography contains scientific, technical, financial, and regulatory information that pertains to DOE environmentalmore » restoration programs. The citations are separated by topic into 16 sections, including (1) DOE Environmental Restoration Program; (2) DOE D&D Program; (3) Nuclear Facilities Decommissioning; (4) DOE Formerly Utilized Sites Remedial Action Programs; (5) NORM-Contaminated Site Restoration; (6) DOE Uranium Mill Tailings Remedial Action Project; (7) Uranium Mill Tailings Management; (8) DOE Site-Wide Remedial Actions; (9) DOE Onsite Remedial Action Projects; (10) Contaminated Site Remedial Actions; (11) DOE Underground Storage Tank Remediation; (12) DOE Technology Development, Demonstration, and Evaluations; (13) Soil Remediation; (14) Groundwater Remediation; (15) Environmental Measurements, Analysis, and Decision-Making; and (16) Environmental Management Issues. Within the 16 sections, the citations are sorted by geographic location. If a geographic location is not specified, the citations are sorted according to the document title. In Part 2 of the report, indexes are provided for author, author affiliation, selected title phrase, selected title word, publication description, geographic location, and keyword.« less

  13. CRISPR/Cas9 for cancer research and therapy.

    PubMed

    Zhan, Tianzuo; Rindtorff, Niklas; Betge, Johannes; Ebert, Matthias P; Boutros, Michael

    2018-04-16

    CRISPR/Cas9 has become a powerful method for making changes to the genome of many organisms. First discovered in bacteria as part of an adaptive immune system, CRISPR/Cas9 and modified versions have found a widespread use to engineer genomes and to activate or to repress the expression of genes. As such, CRISPR/Cas9 promises to accelerate cancer research by providing an efficient technology to dissect mechanisms of tumorigenesis, identify targets for drug development, and possibly arm cells for cell-based therapies. Here, we review current applications of the CRISPR/Cas9 technology for cancer research and therapy. We describe novel Cas9 variants and how they are used in functional genomics to discover novel cancer-specific vulnerabilities. Furthermore, we highlight the impact of CRISPR/Cas9 in generating organoid and mouse models of cancer. Finally, we provide an overview of the first clinical trials that apply CRISPR/Cas9 as a therapeutic approach against cancer. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes.

    PubMed

    Chu, Van Trung; Weber, Timm; Graf, Robin; Sommermann, Thomas; Petsch, Kerstin; Sack, Ulrike; Volchkov, Pavel; Rajewsky, Klaus; Kühn, Ralf

    2016-01-16

    The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic 'safe harbor' site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8-11 kb inserts into Rosa26 of C57BL/6 zygotes. We found that 10-20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9. Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain.

  15. Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system.

    PubMed

    Chen, Xiugui; Lu, Xuke; Shu, Na; Wang, Shuai; Wang, Junjuan; Wang, Delong; Guo, Lixue; Ye, Wuwei

    2017-03-13

    The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H + -pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6-81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome.

  16. Pedestrian and motorists' actions at pedestrian hybrid beacon sites: findings from a pilot study.

    PubMed

    Pulugurtha, Srinivas S; Self, Debbie R

    2015-01-01

    This paper focuses on an analysis of pedestrian and motorists' actions at sites with pedestrian hybrid beacons and assesses their effectiveness in improving the safety of pedestrians. Descriptive and statistical analyses (one-tail two-sample T-test and two-proportion Z-test) were conducted using field data collected during morning and evening peak hours at three study sites in the city of Charlotte, NC, before and after the installation of pedestrian hybrid beacons. Further, an analysis was conducted to assess the change in pedestrian and motorists' actions over time (before the installation; 1 month, 3 months, 6 months, and 12 months after the installation). Results showed an increase in average traffic speed at one of the pedestrian hybrid beacon sites while no specific trends were observed at the other two pedestrian hybrid beacon sites. A decrease in the number of motorists not yielding to pedestrians, pedestrians trapped in the middle of the street, and pedestrian-vehicle conflicts were observed at all the three pedestrian hybrid beacon sites. The installation of pedestrian hybrid beacons did not have a negative effect on pedestrian actions at two out of the three sites. Improvements seem to be relatively more consistent 3 months after the installation of the pedestrian hybrid beacon.

  17. Corrective Action Investigation Plan for Corrective Action Unit 413: Clean Slate II Plutonium Dispersion (TTR) Tonopah Test Range, Nevada, Revision 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick; Burmeister, Mark; Gallo, Patricia

    Corrective Action Unit (CAU) 413 is located on the Tonopah Test Range, which is approximately 130 miles northwest of Las Vegas, Nevada, and approximately 40 miles southeast of Tonopah, Nevada. The CAU 413 site consists of the release of radionuclides to the surface and shallow subsurface from the conduct of the Clean Slate II (CSII) storage–transportation test conducted on May 31, 1963. CAU 413 includes one corrective action site (CAS), TA-23-02CS (Pu Contaminated Soil). The known releases at CAU 413 are the result of the atmospheric deposition of contamination from the 1963 CSII test. The CSII test was a non-nuclearmore » detonation of a nuclear device located inside a reinforced concrete bunker covered with 2 feet of soil. This test dispersed radionuclides, primarily plutonium, on the ground surface. The presence and nature of contamination at CAU 413 will be evaluated based on information collected from a corrective action investigation (CAI). The investigation is based on the data quality objectives (DQOs) developed on June 17, 2015, by representatives of the Nevada Division of Environmental Protection; the U.S. Air Force; and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Field Office. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 413. The CAI will include radiological surveys, geophysical surveys, collection and analyses of soil samples, and assessment of investigation results. The collection of soil samples will be accomplished using both probabilistic and judgmental sampling approaches. To facilitate site investigation and the evaluation of DQO decisions, the releases at CAU 413 have been divided into seven study groups.« less

  18. Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System.

    PubMed

    Cooper, Lauren A; Stringer, Anne M; Wade, Joseph T

    2018-04-17

    In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo , for the type I-E system of Escherichia coli Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5' end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing. IMPORTANCE Many bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61-nucleotide (nt) CRISPR RNAs (crRNAs). The Cascade complex is directed to invading DNA molecules through base pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease, which destroys the invading DNA molecule and promotes acquisition of new immunity elements. We made the first in vivo measurements of Cascade binding to DNA

  19. Advances in therapeutic CRISPR/Cas9 genome editing.

    PubMed

    Savić, Nataša; Schwank, Gerald

    2016-02-01

    Targeted nucleases are widely used as tools for genome editing. Two years ago the clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease was used for the first time, and since then has largely revolutionized the field. The tremendous success of the CRISPR/Cas9 genome editing tool is powered by the ease design principle of the guide RNA that targets Cas9 to the desired DNA locus, and by the high specificity and efficiency of CRISPR/Cas9-generated DNA breaks. Several studies recently used CRISPR/Cas9 to successfully modulate disease-causing alleles in vivo in animal models and ex vivo in somatic and induced pluripotent stem cells, raising hope for therapeutic genome editing in the clinics. In this review, we will summarize and discuss such preclinical CRISPR/Cas9 gene therapy reports. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Recent Advances in Genome Editing Using CRISPR/Cas9.

    PubMed

    Ding, Yuduan; Li, Hong; Chen, Ling-Ling; Xie, Kabin

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 technology, and discuss their implications for plant research. To date, targeted gene knockout using the Cas9/gRNA system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR/Cas9 system can also be used for sequence-specific mutagenesis/integration and transcriptional control of target genes. We also discuss off-target effects and the constraint that the protospacer-adjacent motif (PAM) puts on CRISPR/Cas9 genome engineering. To address these problems, a number of bioinformatic tools are available to help design specific gRNAs, and new Cas9 variants and orthologs with high fidelity and alternative PAM specificities have been engineered. Owing to these recent efforts, the CRISPR/Cas9 system is becoming a revolutionary and flexible tool for genome engineering. Adoption of the CRISPR/Cas9 technology in plant research would enable the investigation of plant biology at an unprecedented depth and create innovative applications in precise crop breeding.

  1. Recent Advances in Genome Editing Using CRISPR/Cas9

    PubMed Central

    Ding, Yuduan; Li, Hong; Chen, Ling-Ling; Xie, Kabin

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 technology, and discuss their implications for plant research. To date, targeted gene knockout using the Cas9/gRNA system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR/Cas9 system can also be used for sequence-specific mutagenesis/integration and transcriptional control of target genes. We also discuss off-target effects and the constraint that the protospacer-adjacent motif (PAM) puts on CRISPR/Cas9 genome engineering. To address these problems, a number of bioinformatic tools are available to help design specific gRNAs, and new Cas9 variants and orthologs with high fidelity and alternative PAM specificities have been engineered. Owing to these recent efforts, the CRISPR/Cas9 system is becoming a revolutionary and flexible tool for genome engineering. Adoption of the CRISPR/Cas9 technology in plant research would enable the investigation of plant biology at an unprecedented depth and create innovative applications in precise crop breeding. PMID:27252719

  2. Assisting Students' Cognitive Strategies with the Use of CAS

    ERIC Educational Resources Information Center

    Sarvari, Csaba; Lavicza, Zsolt; Klincsik, Mihaly

    2010-01-01

    This paper examines various cognitive strategies applied while CAS (Computer Algebra System) are used in undergraduate-level engineering mathematics teaching and learning. We posed some questions in relation to such CAS use: What kind of tools can CAS offer to enhance different cognitive strategies of students? How can the use of CAS widen the…

  3. Production of Purified CasRNPs for Efficacious Genome Editing.

    PubMed

    Lingeman, Emily; Jeans, Chris; Corn, Jacob E

    2017-10-02

    CRISPR-Cas systems have been harnessed as modular genome editing reagents for functional genomics and show promise to cure genetic diseases. Directed by a guide RNA, a Cas effector introduces a double stranded break in DNA and host cell DNA repair leads to the introduction of errors (e.g., to knockout a gene) or a programmed change. Introduction of a Cas effector and guide RNA as a purified Cas ribonucleoprotein complex (CasRNP) has recently emerged as a powerful approach to alter cell types and organisms. Not only does CasRNP editing exhibit increased efficacy and specificity, it avoids optimization and iteration of species-specific factors such as codon usage, promoters, and terminators. CasRNP editing has been rapidly adopted for research use in many contexts and is quickly becoming a popular method to edit primary cells for therapeutic application. This article describes how to make a Cas9 RNP and outlines its use for gene editing in human cells. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  4. Remedial action plan and site design for stabilization of the inactive uranium mill tailings sites at Rifle, Colorado. Volume 2, Appendices D and E: Final report

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Not Available

    1992-02-01

    This appendix assesses the present conditions and data gathered about the two inactive uranium mill tailings sites near Rifle, Colorado, and the designated disposal site six miles north of Rifle in the area of Estes Gulch. It consolidates available engineering, radiological, geotechnical, hydrological, meteorological, and other information pertinent to the design of the Remedial Action Plan (RAP). The data characterize conditions at the mill, tailings, and disposal site so that the Remedial Action Contractor (RAC) may complete final designs for the remedial actions.

  5. A Cas9 transgenic Plasmodium yoelii parasite for efficient gene editing.

    PubMed

    Qian, Pengge; Wang, Xu; Yang, Zhenke; Li, Zhenkui; Gao, Han; Su, Xin-Zhuan; Cui, Huiting; Yuan, Jing

    2018-06-01

    The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium. However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii, we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite (PyCas9ki) of the rodent malaria parasite P. yoelii. The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage. By introducing the plasmid (pYCs) containing only sgRNA and homologous template elements, we successfully achieved both deletion and tagging modifications for different endogenous genes in the genome of PyCas9ki parasite. This cas9-knockin PyCas9ki parasite provides a new platform facilitating gene functions study in the rodent malaria parasite P. yoelii. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Structural and functional insights into the interaction between the Cas family scaffolding protein p130Cas and the focal adhesion-associated protein paxillin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Chi; Miller, Darcie J.; Guibao, Cristina D.

    The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by whichmore » the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ~4.2 μM) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.« less

  7. Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a.

    PubMed

    Swarts, Daan C; van der Oost, John; Jinek, Martin

    2017-04-20

    The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. CRISPR/Cas9-based tools for targeted genome editing and replication control of HBV.

    PubMed

    Peng, Cheng; Lu, Mengji; Yang, Dongliang

    2015-10-01

    Hepatitis B virus (HBV) infection remains a major global health problem because current therapies rarely eliminate HBV infections to achieve a complete cure. A different treatment paradigm to effectively clear HBV infection and eradicate latent viral reservoirs is urgently required. In recent years, the development of a new RNA-guided gene-editing tool, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system, has greatly facilitated site-specific mutagenesis and represents a very promising potential therapeutic tool for diseases, including for eradication of invasive pathogens such as HBV. Here, we review recent advances in the use of CRISPR/Cas9, which is designed to target HBV specific DNA sequences to inhibit HBV replication and to induce viral genome mutation, in cell lines or animal models. Advantages, limitations and possible solutions, and proposed directions for future research are discussed to highlight the opportunities and challenges of CRISPR/Cas9 as a new, potentially curative therapy for chronic hepatitis B infection.

  9. CORRRECTIVE ACTION DECISION DOCUMENT FOR CORRECTIVE ACTION UNIT 427: AREA 3 SEPTIC WASTE SYSTEMS 2 AND 6, TONOPAH TEST RANGE, NEVADA, REVISION 0, JUNE 1998

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    ITLV.

    1998-06-01

    This Corrective Action Decision Document has been prepared for the Area 3 Septic Waste Systems 2 and 6 (Corrective Action Unit 427) in accordance with the Federal Facility Agreement and Consent Order of 1996 (FFACO, 1996). Corrective Action Unit 427 is located at the Tonopah Test Range, Nevada, and is comprised of the following Corrective Action Sites, each an individual septic waste system (DOE/NV, 1996a): Septic Waste System 2 is Corrective Action Site Number 03-05-002-SW02. Septic Waste System 6 is Corrective Action Site Number 03-05-002-SW06. The purpose of this Corrective Action Decision Document is to identify and provide a rationalemore » for the selection of a recommended corrective action alternative for each Corrective Action Site. The scope of this Correction Action Decision Document consists of the following tasks: Develop corrective action objectives. Identify corrective action alternative screening criteria. Develop corrective action alternatives. Perform detailed and comparative evaluations of the corrective action alternatives in relation to the corrective action objectives and screening criteria. Recommend and justify a preferred corrective action alternative for each CAS. From November 1997 through January 1998, a corrective action investigation was performed as set forth in the Corrective Action Investigation Plan for Corrective Action Unit No. 427: Area 3 Septic Waste System Numbers 2 and 6, Tonopah Test Range, Nevada (DOE/NV, 1997b). Details can be found in Appendix A of this document. The results indicated that contamination is present in some portions of the CAU and not in others as described in Table ES-1 and shown in Figure A.2-2 of Appendix A. Based on the potential exposure pathways, the following corrective action objectives have been identified for Corrective Action Unit 427: Prevent or mitigate human exposure to subsurface soils containing TPH at concentrations greater than 100 milligrams per kilogram (NAC, 1996b). Close Septic Tank

  10. The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit.

    PubMed

    Murugan, Karthik; Babu, Kesavan; Sundaresan, Ramya; Rajan, Rakhi; Sashital, Dipali G

    2017-10-05

    CRISPR-Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR-Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR-Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR-Cas systems. This diversity has enabled further expansion of CRISPR-Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR-Cas systems. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Subtyping of the Legionella pneumophila "Ulm" outbreak strain using the CRISPR-Cas system.

    PubMed

    Lück, Christian; Brzuszkiewicz, Elzbieta; Rydzewski, Kerstin; Koshkolda, Tetyana; Sarnow, Katharina; Essig, Andreas; Heuner, Klaus

    2015-12-01

    In 2009/2010 an outbreak of Legionnaires' disease with 64 cases including four fatalities took place in the city of Ulm/Neu-Ulm in Germany. L. pneumophila serogroup 1, mAb type Knoxville, sequence type (ST) 62 was identified as the epidemic strain. This strain was isolated from eight patients and from a cooling tower in the city of Ulm. Based on whole genome sequencing data from one patient strain, we identified an Lvh type IV secretion system containing a CRISPR-Cas system. The CRISPR sequence contains 38 spacer DNA sequences. We used these variable DNA spacers to further subtype the outbreak strain as well as six epidemiologically unrelated strains of CRISPR-Cas positive ST62 strains isolated at various regions in Germany. The first 12 spacer DNAs of eight patient isolates and three environmental isolates from the suspected source of infection were analyzed and found to be identical. Spacer DNAs were identified in further six epidemiologically unrelated patient isolates of L. pneumophila of ST62 in addition to the 12 "core" spacers. The presence of new spacer DNAs at the 5' site downstream of the first repeat indicates that these CRISPR-Cas systems seem to be functional. PCR analysis revealed that not all L. pneumophila sg1 ST62 strains investigated exhibited a CRISPR-Cas system. In addition, we could demonstrate that the CRISPR-Cas system is localized on a genomic island (LpuGI-Lvh) which can be excised from the chromosome and therefore may be transferable horizontally to other L. pneumophila strains. Copyright © 2015 Elsevier GmbH. All rights reserved.

  12. Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9.

    PubMed

    Chung, Mu-En; Yeh, I-Hsin; Sung, Li-Yu; Wu, Meng-Ying; Chao, Yun-Peng; Ng, I-Son; Hu, Yu-Chen

    2017-01-01

    Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA-guided system which enables site-specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9-triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ-Red proteins and linear dsDNA, CRISPR/Cas9-induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9-assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172-183. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Nuclear facility decommissioning and site remedial actions: A selected bibliography, Volume 13: Part 1, Main text

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Goins, L.F.; Webb, J.R.; Cravens, C.D.

    1992-09-01

    This publication contains 1035 abstracted references on environmental restoration, nuclear facility decommissioning, uranium mill tailings management, and site remedial actions. These citations constitute the thirteenth in a series of reports prepared annually for the US Department of Energy (DOE) Environmental Restoration programs. Citations to foreign and domestic literature of all types. There are 13 major sections of the publication, including: (1) DOE Decontamination and Decommissioning Program; (2) Nuclear Facilities Decommissioning; (3) DOE Formerly Utilized Sites Remedial Action Program; (4) DOE Uranium Mill Tailings Remedial Action Project; (5) Uranium Mill Tailings Management; (6) DOE Environmental Restoration Program; (7) DOE Site-Specific Remedialmore » Actions; (8) Contaminated Site Restoration; (9) Remediation of Contaminated Soil and Groundwater; (10) Environmental Data Measurements, Management, and Evaluation; (11) Remedial Action Assessment and Decision-Making; (12) Technology Development and Evaluation; and (13) Environmental and Waste Management Issues. Bibliographic references are arranged in nine subject categories by geographic location and then alphabetically by first author, corporate affiliation, or publication title. Indexes are provided for author, corporate affiliation, title word, publication description, geographic location, subject category, and key word.« less

  14. Closure Report for Corrective Action Unit 356: Mud Pits and Disposal Sites, Nevada Test Site, Nevada with Errata Sheet

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NNSA /NV

    2002-11-12

    This Closure Report (CR) has been prepared for Corrective Action Unit (CAU) 356, Mud Pits and Disposal Sites, in accordance with the Federal Facility Agreement and Consent Order. This CAU is located in Areas 3 and 20 of the Nevada Test Site (NTS) approximately 65 miles northwest of Las Vegas, Nevada. Corrective Action Unit 356 consists of seven Corrective Action Sites (CASs): 03-04-01, Area 3 Change House Septic System; 03-09-01, Mud Pit Spill Over; 03-09-03, Mud Pit; 03-09-04, Mud Pit; 03-09-05, Mud Pit; 20-16-01, Landfill; and 20-22-21, Drums. This CR identifies and rationalizes the U.S. Department of Energy (DOE), Nationalmore » Nuclear Security Administration Nevada Operations Office's (NNSA/NV's) recommendation that no further corrective action and closure in place is deemed necessary for CAU 356. This recommendation is based on the results of field investigation/closure activities conducted November 20, 2001, through January 3, 2002, and March 11 to 14, 2002. These activities were conducted in accordance with the Streamlined Approach for Environmental Restoration Plan (SAFER) for CAU 356. For CASs 03-09-01, 03-09-03, 20-16-01, and 22-20-21, analytes detected in soil during the corrective action investigation were evaluated against Preliminary Action Levels (PALs) and it was determined that no Contaminants of Concern (COCs) were present. Therefore, no further action is necessary for the soil at these CASs. For CASs 03-04-01, 03-09-04, and 03-09-05, analytes detected in soil during the corrective action investigation were evaluated against PALs and identifies total petroleum hydrocarbons (TPHs) and radionuclides (i.e., americium-241 and/or plutonium 239/240) as COCs. The nature, extent, and concentration of the TPH and radionuclide COCs were bounded by sampling and shown to be relatively immobile. Therefore, closure in place is recommended for these CASs in CAU 356. Further, use restrictions are not required at this CAU beyond the NTS use restrictions

  15. Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3).

    PubMed

    Mulepati, Sabin; Bailey, Scott

    2011-09-09

    RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPR-associated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the single-stranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system.

  16. CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum.

    PubMed

    Pohl, C; Kiel, J A K W; Driessen, A J M; Bovenberg, R A L; Nygård, Y

    2016-07-15

    CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.

  17. New Jersey state information handbook: Formerly Utilized Sites Remedial Action Program

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    None

    Under the implied authority of the Atomic Energy Act of 1954, as amended, radiological surveys and research work has been conducted to determine radiological conditions at former MED/AEC sites. As of this time, 31 sites in 13 states have been identified that require or may require remedial action. This volume is one of a series produced under contract with DOE, Office of Nuclear Waste Management, by POLITECH CORPORATION to develop a legislative and regulatory data base to assist the FUSRAP management in addressing the institutional and socioeconomic issues involved in carrying out the Remedial Action Program. This Information Handbook seriesmore » contains information about all relevant government agencies at the Federal and state levels, the pertinent programs they administer, each affected state legislature, and current Federal and state legislative and regulatory initiatives. This volume is a compilation of information about the state of New Jersey. It contains: a description of the state executive branch structure; a summary of relevant state statutes and regulations; a description of the structure of the state legislature, identification of the officers and committee chairmen, and a summary of recent relevant legislative action; and the full text of relevant statutes and regulations. The loose-leaf format used in these volumes will allow the material to be updated periodically as the Remedial Action Program progresses.« less

  18. Recent Advances in CRISPR-Cas9 Genome Editing Technology for Biological and Biomedical Investigations.

    PubMed

    Singh, Vijai; Gohil, Nisarg; Ramírez García, Robert; Braddick, Darren; Fofié, Christian Kuete

    2018-01-01

    The Type II CRISPR-Cas9 system is a simple, efficient, and versatile tool for targeted genome editing in a wide range of organisms and cell types. It continues to gain more scientific interest and has established itself as an extremely powerful technology within our synthetic biology toolkit. It works upon a targeted site and generates a double strand breaks that become repaired by either the NHEJ or the HDR pathway, modifying or permanently replacing the genomic target sequences of interest. These can include viral targets, single-mutation genetic diseases, and multiple-site corrections for wide scale disease states, offering the potential to manage and cure some of mankind's most persistent biomedical menaces. Here, we present the developing progress and future potential of CRISPR-Cas9 in biological and biomedical investigations, toward numerous therapeutic, biomedical, and biotechnological applications, as well as some of the challenges within. J. Cell. Biochem. 119: 81-94, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. Partial DNA-guided Cas9 enables genome editing with reduced off-target activity

    PubMed Central

    Yin, Hao; Song, Chun-Qing; Suresh, Sneha; Kwan, Suet-Yan; Wu, Qiongqiong; Walsh, Stephen; Ding, Junmei; Bogorad, Roman L; Zhu, Lihua Julie; Wolfe, Scot A; Koteliansky, Victor; Xue, Wen; Langer, Robert; Anderson, Daniel G

    2018-01-01

    CRISPR–Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites. Using the structure of the Cas9–sgRNA complex as a guide, the majority of the 3′ end of crRNA can be replaced with DNA nucleotide, and the 5 - and 3′-DNA-replaced crRNA enables efficient genome editing. Cas9 guided by a DNA–RNA chimera may provide a generalized strategy to reduce both the cost and the off-target genome editing in human cells. PMID:29377001

  20. Development and Potential Applications of CRISPR-Cas9 Genome Editing Technology in Sarcoma

    PubMed Central

    Liu, Tang; Shen, Jacson K.; Li, Zhihong; Choy, Edwin; Hornicek, Francis J.; Duan, Zhenfeng

    2016-01-01

    Sarcomas include some of the most aggressive tumors and typically respond poorly to chemotherapy. In recent years, specific gene fusion/mutations and gene over-expression/activation have been shown to drive sarcoma pathogenesis and development. These emerging genomic alterations may provide targets for novel therapeutic strategies and have the potential to transform sarcoma patient care. The RNA-guided nuclease CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein-9 nuclease) is a convenient and versatile platform for site-specific genome editing and epigenome targeted modulation. Given that sarcoma is believed to develop as a result of genetic alterations in mesenchymal progenitor/stem cells, CRISPR-Cas9 genome editing technologies hold extensive application potentials in sarcoma models and therapies. We review the development and mechanisms of the CRISPR-Cas9 system in genome editing and introduce its application in sarcoma research and potential therapy in clinic. Additionally, we propose future directions and discuss the challenges faced with these applications, providing concise and enlightening information for readers interested in this area. PMID:26806808

  1. Development and potential applications of CRISPR-Cas9 genome editing technology in sarcoma.

    PubMed

    Liu, Tang; Shen, Jacson K; Li, Zhihong; Choy, Edwin; Hornicek, Francis J; Duan, Zhenfeng

    2016-04-01

    Sarcomas include some of the most aggressive tumors and typically respond poorly to chemotherapy. In recent years, specific gene fusion/mutations and gene over-expression/activation have been shown to drive sarcoma pathogenesis and development. These emerging genomic alterations may provide targets for novel therapeutic strategies and have the potential to transform sarcoma patient care. The RNA-guided nuclease CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein-9 nuclease) is a convenient and versatile platform for site-specific genome editing and epigenome targeted modulation. Given that sarcoma is believed to develop as a result of genetic alterations in mesenchymal progenitor/stem cells, CRISPR-Cas9 genome editing technologies hold extensive application potentials in sarcoma models and therapies. We review the development and mechanisms of the CRISPR-Cas9 system in genome editing and introduce its application in sarcoma research and potential therapy in clinic. Additionally, we propose future directions and discuss the challenges faced with these applications, providing concise and enlightening information for readers interested in this area. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Application of CRISPR/Cas9 in plant biology.

    PubMed

    Liu, Xuan; Wu, Surui; Xu, Jiao; Sui, Chun; Wei, Jianhe

    2017-05-01

    The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system was first identified in bacteria and archaea and can degrade exogenous substrates. It was developed as a gene editing technology in 2013. Over the subsequent years, it has received extensive attention owing to its easy manipulation, high efficiency, and wide application in gene mutation and transcriptional regulation in mammals and plants. The process of CRISPR/Cas is optimized constantly and its application has also expanded dramatically. Therefore, CRISPR/Cas is considered a revolutionary technology in plant biology. Here, we introduce the mechanism of the type II CRISPR/Cas called CRISPR/Cas9, update its recent advances in various applications in plants, and discuss its future prospects to provide an argument for its use in the study of medicinal plants.

  3. indCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events.

    PubMed

    Hodgens, Charles; Nimchuk, Zachary L; Kieber, Joseph J

    2017-01-01

    Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/.

  4. Generation of a Knockout Mouse Embryonic Stem Cell Line Using a Paired CRISPR/Cas9 Genome Engineering Tool.

    PubMed

    Wettstein, Rahel; Bodak, Maxime; Ciaudo, Constance

    2016-01-01

    CRISPR/Cas9, originally discovered as a bacterial immune system, has recently been engineered into the latest tool to successfully introduce site-specific mutations in a variety of different organisms. Composed only of the Cas9 protein as well as one engineered guide RNA for its functionality, this system is much less complex in its setup and easier to handle than other guided nucleases such as Zinc-finger nucleases or TALENs.Here, we describe the simultaneous transfection of two paired CRISPR sgRNAs-Cas9 plasmids, in mouse embryonic stem cells (mESCs), resulting in the knockout of the selected target gene. Together with a four primer-evaluation system, it poses an efficient way to generate new independent knockout mouse embryonic stem cell lines.

  5. Genome engineering in ophthalmology: Application of CRISPR/Cas to the treatment of eye disease.

    PubMed

    Hung, Sandy S C; McCaughey, Tristan; Swann, Olivia; Pébay, Alice; Hewitt, Alex W

    2016-07-01

    The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated protein (Cas) system has enabled an accurate and efficient means to edit the human genome. Rapid advances in this technology could results in imminent clinical application, and with favourable anatomical and immunological profiles, ophthalmic disease will be at the forefront of such work. There have been a number of breakthroughs improving the specificity and efficacy of CRISPR/Cas-mediated genome editing. Similarly, better methods to identify off-target cleavage sites have also been developed. With the impending clinical utility of CRISPR/Cas technology, complex ethical issues related to the regulation and management of the precise applications of human gene editing must be considered. This review discusses the current progress and recent breakthroughs in CRISPR/Cas-based gene engineering, and outlines some of the technical issues that must be addressed before gene correction, be it in vivo or in vitro, is integrated into ophthalmic care. We outline a clinical pipeline for CRISPR-based treatments of inherited eye diseases and provide an overview of the important ethical implications of gene editing and how these may influence the future of this technology. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.

    PubMed

    Rouet, Romain; Thuma, Benjamin A; Roy, Marc D; Lintner, Nathanael G; Rubitski, David M; Finley, James E; Wisniewska, Hanna M; Mendonsa, Rima; Hirsh, Ariana; de Oñate, Lorena; Compte Barrón, Joan; McLellan, Thomas J; Bellenger, Justin; Feng, Xidong; Varghese, Alison; Chrunyk, Boris A; Borzilleri, Kris; Hesp, Kevin D; Zhou, Kaihong; Ma, Nannan; Tu, Meihua; Dullea, Robert; McClure, Kim F; Wilson, Ross C; Liras, Spiros; Mascitti, Vincent; Doudna, Jennifer A

    2018-05-30

    CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

  7. CRISPR-Cas Genome Surgery in Ophthalmology

    PubMed Central

    DiCarlo, James E.; Sengillo, Jesse D.; Justus, Sally; Cabral, Thiago; Tsang, Stephen H.; Mahajan, Vinit B.

    2017-01-01

    Genetic disease affecting vision can significantly impact patient quality of life. Gene therapy seeks to slow the progression of these diseases by treating the underlying etiology at the level of the genome. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (Cas) represent powerful tools for studying diseases through the creation of model organisms generated by targeted modification and by the correction of disease mutations for therapeutic purposes. CRISPR-Cas systems have been applied successfully to the visual sciences and study of ophthalmic disease – from the modification of zebrafish and mammalian models of eye development and disease, to the correction of pathogenic mutations in patient-derived stem cells. Recent advances in CRISPR-Cas delivery and optimization boast improved functionality that continues to enhance genome-engineering applications in the eye. This review provides a synopsis of the recent implementations of CRISPR-Cas tools in the field of ophthalmology. PMID:28573077

  8. Editing plants for virus resistance using CRISPR-Cas.

    PubMed

    Green, J C; Hu, J S

    This minireview summarizes recent advancements using the clustered regularly interspaced palindromic repeats-associated nuclease systems (CRISPR-Cas) derived from prokaryotes to breed plants resistant to DNA and RNA viruses. The CRISPR-Cas system represents a powerful tool able to edit and insert novel traits into plants precisely at chosen loci offering enormous advantages to classical breeding. Approaches to engineering plant virus resistance in both transgenic and non-transgenic plants are discussed. Iterations of the CRISPR-Cas system, FnCas9 and C2c2 capable of editing RNA in eukaryotic cells offer a particular advantage for providing resistance to RNA viruses which represent the great majority of known plant viruses. Scientists have obtained conflicting results using gene silencing technology to produce transgenic plants resistant to geminiviruses. CRISPR-Cas systems engineered in plants to target geminiviruses have consistently reduced virus accumulation providing increased resistance to virus infection. CRISPR-Cas may provide novel and reliable approaches to control geminiviruses and other ssDNA viruses such as Banana bunchy top virus (BBTV).

  9. Scarless Cas9 Assisted Recombineering (no-SCAR) in Escherichia coli, an Easy-to-Use System for Genome Editing.

    PubMed

    Reisch, Christopher R; Prather, Kristala L J

    2017-01-05

    The discovery and development of genome editing systems that leverage the site-specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a "guide" RNA to enable the Cas9 nuclease to make a double-strand break at a particular genome locus, which is repaired by non-homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration of the target genome can also be generated by supplying a DNA template in vivo with a desired mutation, which is incorporated by homology-directed repair. However, E. coli lacks robust systems for double-strand break repair. Thus, in contrast to eukaryotes, targeting E. coli chromosomal DNA with Cas9 causes cell death. However, Cas9-mediated killing of bacteria can be exploited to select against cells with a specified genotype within a mixed population. In combination with the well described λ-Red system for recombination in E. coli, we created a highly efficient system for marker-free and scarless genome editing. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  10. Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system

    PubMed Central

    Chen, Xiugui; Lu, Xuke; Shu, Na; Wang, Shuai; Wang, Junjuan; Wang, Delong; Guo, Lixue; Ye, Wuwei

    2017-01-01

    The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H+-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6–81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome. PMID:28287154

  11. Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity.

    PubMed

    Seamon, Kyle J; Light, Yooli K; Saada, Edwin A; Schoeniger, Joseph S; Harmon, Brooke

    2018-06-05

    The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.

  12. Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Seamon, Kyle Jeffrey; Light, Yooli Kim; Saada, Edwin A.

    Here, the RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate itsmore » utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.« less

  13. Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

    DOE PAGES

    Seamon, Kyle Jeffrey; Light, Yooli Kim; Saada, Edwin A.; ...

    2018-05-14

    Here, the RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate itsmore » utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.« less

  14. Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep.

    PubMed

    Wang, Xiaolong; Liu, Jing; Niu, Yiyuan; Li, Yan; Zhou, Shiwei; Li, Chao; Ma, Baohua; Kou, Qifang; Petersen, Bjoern; Sonstegard, Tad; Huang, Xingxu; Jiang, Yu; Chen, Yulin

    2018-05-25

    The simplicity of the CRISPR/Cas9 system has enabled its widespread applications in generating animal models, functional genomic screening and in treating genetic and infectious diseases. However, unintended mutations produced by off-target CRISPR/Cas9 nuclease activity may lead to negative consequences. Especially, a very recent study found that gene editing can introduce hundreds of unintended mutations into the genome, and have attracted wide attention. To address the off-target concerns, urgent characterization of the CRISPR/Cas9-mediated off-target mutagenesis is highly anticipated. Here we took advantage of our previously generated gene-edited sheep and performed family trio-based whole genome sequencing which is capable of discriminating variants in the edited progenies that are inherited, naturally generated, or induced by genetic modification. Three family trios were re-sequenced at a high average depth of genomic coverage (~ 25.8×). After developing a pipeline to comprehensively analyze the sequence data for de novo single nucleotide variants, indels and structural variations from the genome; we only found a single unintended event in the form of a 2.4 kb inversion induced by site-specific double-strand breaks between two sgRNA targeting sites at the MSTN locus with a low incidence. We provide the first report on the fidelity of CRISPR-based modification for sheep genomes targeted simultaneously for gene breaks at three coding sequence locations. The trio-based sequencing approach revealed almost negligible off-target modifications, providing timely evidences of the safe application of genome editing in vivo with CRISPR/Cas9.

  15. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation.

    PubMed

    Zhang, Hui; Zhang, Jinshan; Wei, Pengliang; Zhang, Botao; Gou, Feng; Feng, Zhengyan; Mao, Yanfei; Yang, Lan; Zhang, Heng; Xu, Nanfei; Zhu, Jian-Kang

    2014-08-01

    The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9-induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9-induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large-scale off-targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off-target sites of a large number of T0 plants, low-frequency mutations were found in only one off-target site where the sequence had 1-bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  16. Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems.

    PubMed

    Yasue, Akihiro; Mitsui, Silvia Naomi; Watanabe, Takahito; Sakuma, Tetsushi; Oyadomari, Seiichi; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro; Tanaka, Eiji

    2014-07-16

    Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice.

  17. Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans

    PubMed Central

    Paix, Alexandre; Wang, Yuemeng; Smith, Harold E.; Lee, Chih-Yung S.; Calidas, Deepika; Lu, Tu; Smith, Jarrett; Schmidt, Helen; Krause, Michael W.; Seydoux, Geraldine

    2014-01-01

    Homology-directed repair (HDR) of double-strand DNA breaks is a promising method for genome editing, but is thought to be less efficient than error-prone nonhomologous end joining in most cell types. We have investigated HDR of double-strand breaks induced by CRISPR-associated protein 9 (Cas9) in Caenorhabditis elegans. We find that HDR is very robust in the C. elegans germline. Linear repair templates with short (∼30–60 bases) homology arms support the integration of base and gene-sized edits with high efficiency, bypassing the need for selection. Based on these findings, we developed a systematic method to mutate, tag, or delete any gene in the C. elegans genome without the use of co-integrated markers or long homology arms. We generated 23 unique edits at 11 genes, including premature stops, whole-gene deletions, and protein fusions to antigenic peptides and GFP. Whole-genome sequencing of five edited strains revealed the presence of passenger variants, but no mutations at predicted off-target sites. The method is scalable for multi-gene editing projects and could be applied to other animals with an accessible germline. PMID:25249454

  18. Genome Editing with CRISPR-Cas9: Can It Get Any Better?

    PubMed Central

    Haeussler, Maximilian; Concordet, Jean-Paul

    2017-01-01

    The CRISPR-Cas revolution is taking place in virtually all fields of life sciences. Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient, relying only on the design of a synthetic single guide RNA (sgRNA) and its co-expression with Cas9. Here, we review the progress in the design of sgRNA from the original dual RNA guide for S. pyogenes and Staphylococcus aureus Cas9 (SpCas9 and SaCas9). New assays for genome-wide identification of off-targets have provided important insights into the issue of cleavage specificity in vivo. At the same time, the on-target activity of thousands of guides has been determined. These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences. It appears that for most basic research applications, cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions. Moreover, recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing. Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA, several variants of SpCas9 have recently been engineered, either with novel protospacer adjacent motifs (PAMs) or with drastically reduced off-targets. Novel Cas9 and Cas9-like proteins called Cpf1 have also been characterized from other bacteria and will benefit from the insights obtained from SpCas9. Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage. PMID:27210042

  19. Genome Editing with CRISPR-Cas9: Can It Get Any Better?

    PubMed

    Haeussler, Maximilian; Concordet, Jean-Paul

    2016-05-20

    The CRISPR-Cas revolution is taking place in virtually all fields of life sciences. Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient, relying only on the design of a synthetic single guide RNA (sgRNA) and its co-expression with Cas9. Here, we review the progress in the design of sgRNA from the original dual RNA guide for S. pyogenes and Staphylococcus aureus Cas9 (SpCas9 and SaCas9). New assays for genome-wide identification of off-targets have provided important insights into the issue of cleavage specificity in vivo. At the same time, the on-target activity of thousands of guides has been determined. These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences. It appears that for most basic research applications, cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions. Moreover, recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing. Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA, several variants of SpCas9 have recently been engineered, either with novel protospacer adjacent motifs (PAMs) or with drastically reduced off-targets. Novel Cas9 and Cas9-like proteins called Cpf1 have also been characterized from other bacteria and will benefit from the insights obtained from SpCas9. Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  20. Multidrug-resistant enterococci lack CRISPR-cas.

    PubMed

    Palmer, Kelli L; Gilmore, Michael S

    2010-10-12

    Clustered, regularly interspaced short palindromic repeats (CRISPR) provide bacteria and archaea with sequence-specific, acquired defense against plasmids and phage. Because mobile elements constitute up to 25% of the genome of multidrug-resistant (MDR) enterococci, it was of interest to examine the codistribution of CRISPR and acquired antibiotic resistance in enterococcal lineages. A database was built from 16 Enterococcus faecalis draft genome sequences to identify commonalities and polymorphisms in the location and content of CRISPR loci. With this data set, we were able to detect identities between CRISPR spacers and sequences from mobile elements, including pheromone-responsive plasmids and phage, suggesting that CRISPR regulates the flux of these elements through the E. faecalis species. Based on conserved locations of CRISPR and CRISPR-cas loci and the discovery of a new CRISPR locus with associated functional genes, CRISPR3-cas, we screened additional E. faecalis strains for CRISPR content, including isolates predating the use of antibiotics. We found a highly significant inverse correlation between the presence of a CRISPR-cas locus and acquired antibiotic resistance in E. faecalis, and examination of an additional eight E. faecium genomes yielded similar results for that species. A mechanism for CRISPR-cas loss in E. faecalis was identified. The inverse relationship between CRISPR-cas and antibiotic resistance suggests that antibiotic use inadvertently selects for enterococcal strains with compromised genome defense.

  1. La tuberculose cutanée: observation de six cas confirmés au CHU Souro SANOU (CHUSS) de Bobo-Dioulasso (Burkina Faso)

    PubMed Central

    Andonaba, Jean Baptiste; Barro-Traoré, Fatou; Yaméogo, Téné; Diallo, Boukary; Korsaga-Somé, Nina; Traoré, Adama

    2013-01-01

    La localisation cutanée de la maladie tuberculeuse demeure une forme rare et représente seulement 2,1% des localisations. L'objet de cette étude est de rapporter le profil épidémiologique, anatomoclinique et évolutif des cas de tuberculose ganglio-cutanée diagnostiqués dans un CHU au Burkina Faso. La fréquence de la tuberculose cutanée est très faible au CHUSS. Six cas ont été diagnostiqués entre 2004 et 2010, soit une fréquence de un cas par an. La durée d’évolution des cas allait de deux jusqu’à dix ans avant leur diagnostic. Les lésions observées étaient: trois scrofulodermes, trois gommes, une tuberculose testiculaire associée à un mal de Pott, un cas de polyadénopathies et des cicatrices atropho-rétractiles dans la plupart des cas. Sur le plan anatomopathologique, des granulomes tuberculoïdes ont été mis en évidence dans tous les cas avec une forte réaction tuberculinique à l'IDR. Sous antituberculeux pendant six mois, l’évolution a été bonne dans tous les cas mais au prix de séquelles cutanées cicatricielles inesthétiques. Son ampleur reste peut-être encore méconnue. Le renforcement du plateau technique du CHU et une bonne collaboration interdisciplinaire contribuerait à un meilleur diagnostic et prise en charge de cette affection. PMID:24648863

  2. The role of Cas8 in type I CRISPR interference.

    PubMed

    Cass, Simon D B; Haas, Karina A; Stoll, Britta; Alkhnbashi, Omer S; Sharma, Kundan; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita; Bolt, Edward L

    2015-05-05

    CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use 'cascade' [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5-Cas7-crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA. © 2015 Authors.

  3. The trace element chemistry of CaS in enstatite chondrites and some implications regarding its origin

    NASA Technical Reports Server (NTRS)

    Larimer, John W.; Ganapathy, R.

    1987-01-01

    The trace element distribution in oldhamite (CaS) extracted from enstatite chondrites was determined by INAA. Prior to extraction, the petrologic setting of the grains was studied microscopically, and their minor element contents determined by microprobe analysis; samples that displayed a wide range of minor element contents were selected for detailed elementary analysis. Those samples of CaS suspected to be more primitive on the basis of their minor element and petrologic siting contain the entire inventory of the host meteorite's light REE (LREE) and Eu, plus 30-50 percent of the heavy-REE inventory. In less primitive samples, the LREE are less enriched although Eu remains highly concentrated. Several other elements, including lithophiles and chalcophiles, are most enriched in the most primitive CaS. It is suggested that oldhamite played a key role in the redistribution of these elements during the metamorphism and evolution of enstatite-rich material.

  4. Exploiting CRISPR-Cas to manipulate Enterococcus faecalis populations.

    PubMed

    Hullahalli, Karthik; Rodrigues, Marinelle; Palmer, Kelli L

    2017-06-23

    CRISPR-Cas provides a barrier to horizontal gene transfer in prokaryotes. It was previously observed that functional CRISPR-Cas systems are absent from multidrug-resistant (MDR) Enterococcus faecalis , which only possess an orphan CRISPR locus, termed CRISPR2, lacking cas genes. Here, we investigate how the interplay between CRISPR-Cas genome defense and antibiotic selection for mobile genetic elements shapes in vitro E. faecalis populations. We demonstrate that CRISPR2 can be reactivated for genome defense in MDR strains. Interestingly, we observe that E. faecalis transiently maintains CRISPR targets despite active CRISPR-Cas systems. Subsequently, if selection for the CRISPR target is present, toxic CRISPR spacers are lost over time, while in the absence of selection, CRISPR targets are lost over time. We find that forced maintenance of CRISPR targets induces a fitness cost that can be exploited to alter heterogeneous E. faecalis populations.

  5. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    .... 9903.201-2 Section 9903.201-2 Federal Acquisition Regulations System COST ACCOUNTING STANDARDS BOARD... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full... net CAS-covered awards during its preceding cost accounting period. (b) Modified coverage. (1...

  6. NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer.

    PubMed

    Wallez, Yann; Mace, Peter D; Pasquale, Elena B; Riedl, Stefan J

    2012-05-01

    The CAS (CRK-associated substrate) family of adaptor proteins comprises 4 members, which share a conserved modular domain structure that enables multiple protein-protein interactions, leading to the assembly of intracellular signaling platforms. Besides their physiological role in signal transduction downstream of a variety of cell surface receptors, CAS proteins are also critical for oncogenic transformation and cancer cell malignancy through associations with a variety of regulatory proteins and downstream effectors. Among the regulatory partners, the 3 recently identified adaptor proteins constituting the NSP (novel SH2-containing protein) family avidly bind to the conserved carboxy-terminal focal adhesion-targeting (FAT) domain of CAS proteins. NSP proteins use an anomalous nucleotide exchange factor domain that lacks catalytic activity to form NSP-CAS signaling modules. Additionally, the NSP SH2 domain can link NSP-CAS signaling assemblies to tyrosine-phosphorylated cell surface receptors. NSP proteins can potentiate CAS function by affecting key CAS attributes such as expression levels, phosphorylation state, and subcellular localization, leading to effects on cell adhesion, migration, and invasion as well as cell growth. The consequences of these activities are well exemplified by the role that members of both families play in promoting breast cancer cell invasiveness and resistance to antiestrogens. In this review, we discuss the intriguing interplay between the NSP and CAS families, with a particular focus on cancer signaling networks.

  7. Control of gene expression by CRISPR-Cas systems

    PubMed Central

    2013-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated cas (CRISPR-associated) genes provide adaptive immunity against viruses (phages) and other mobile genetic elements in bacteria and archaea. While most of the early work has largely been dominated by examples of CRISPR-Cas systems directing the cleavage of phage or plasmid DNA, recent studies have revealed a more complex landscape where CRISPR-Cas loci might be involved in gene regulation. In this review, we summarize the role of these loci in the regulation of gene expression as well as the recent development of synthetic gene regulation using engineered CRISPR-Cas systems. PMID:24273648

  8. Generation of Myostatin Gene-Edited Channel Catfish (Ictalurus punctatus) via Zygote Injection of CRISPR/Cas9 System.

    PubMed

    Khalil, Karim; Elayat, Medhat; Khalifa, Elsayed; Daghash, Samer; Elaswad, Ahmed; Miller, Michael; Abdelrahman, Hisham; Ye, Zhi; Odin, Ramjie; Drescher, David; Vo, Khoi; Gosh, Kamal; Bugg, William; Robinson, Dalton; Dunham, Rex

    2017-08-04

    The myostatin (MSTN) gene is important because of its role in regulation of skeletal muscle growth in all vertebrates. In this study, CRISPR/Cas9 was utilized to successfully target the channel catfish, Ictalurus punctatus, muscle suppressor gene MSTN. CRISPR/Cas9 induced high rates (88-100%) of mutagenesis in the target protein-encoding sites of MSTN. MSTN-edited fry had more muscle cells (p < 0.001) than controls, and the mean body weight of gene-edited fry increased by 29.7%. The nucleic acid alignment of the mutated sequences against the wild-type sequence revealed multiple insertions and deletions. These results demonstrate that CRISPR/Cas9 is a highly efficient tool for editing the channel catfish genome, and opens ways for facilitating channel catfish genetic enhancement and functional genomics. This approach may produce growth-enhanced channel catfish and increase productivity.

  9. Closure Report for Corrective Action Unit 568: Area 3 Plutonium Dispersion Sites Nevada National Security Site, Nevada, Revision 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matthews, Patrick

    The purpose of this CR is to provide documentation and justification that no further corrective action is needed for the closure of CAU 568 based on the implementation of corrective actions. This includes a description of closure activities that were performed and an evaluation of the verification data. The CAP (NNSA/NFO, 2016a) and ROTC-1 (NNSA/NFO, 2016c) provide information relating to the selection of CAAs and the reasoning behind their selection. The CADD (NNSA/NFO, 2015) identifies the release sites that require additional corrective action and presents information supporting the selection of CAAs.

  10. Cas9 Variants Expand the Target Repertoire in Caenorhabditis elegans

    PubMed Central

    Bell, Ryan T.; Fu, Becky X. H.; Fire, Andrew Z.

    2016-01-01

    The proliferation of CRISPR/Cas9-based methods in Caenorhabditis elegans has enabled efficient genome editing and precise genomic tethering of Cas9 fusion proteins. Experimental designs using CRISPR/Cas9 are currently limited by the need for a protospacer adjacent motif (PAM) in the target with the sequence NGG. Here we report the characterization of two modified Cas9 proteins in C. elegans that recognize NGA and NGCG PAMs. We found that each variant could stimulate homologous recombination with a donor template at multiple loci and that PAM specificity was comparable to that of wild-type Cas9. To directly compare effectiveness, we used CRISPR/Cas9 genome editing to generate a set of assay strains with a common single-guide RNA (sgRNA) target sequence, but that differ in the juxtaposed PAM (NGG, NGA, or NGCG). In this controlled setting, we determined that the NGA PAM Cas9 variant can be as effective as wild-type Cas9. We similarly edited a genomic target to study the influence of the base following the NGA PAM. Using four strains with four NGAN PAMs differing only at the fourth position and adjacent to the same sgRNA target, we observed that efficient homologous replacement was attainable with any base in the fourth position, with an NGAG PAM being the most effective. In addition to demonstrating the utility of two Cas9 mutants in C. elegans and providing reagents that permit CRISPR/Cas9 experiments with fewer restrictions on potential targets, we established a means to benchmark the efficiency of different Cas9::PAM combinations that avoids variations owing to differences in the sgRNA sequence. PMID:26680661

  11. Functional Insights Revealed by the Kinetic Mechanism of CRISPR/Cas9.

    PubMed

    Raper, Austin T; Stephenson, Anthony A; Suo, Zucai

    2018-02-28

    The discovery of prokaryotic adaptive immunity prompted widespread use of the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) endonuclease Cas9 for genetic engineering. However, its kinetic mechanism remains undefined, and details of DNA cleavage are poorly characterized. Here, we establish a kinetic mechanism of Streptococcus pyogenes Cas9 from guide-RNA binding through DNA cleavage and product release. Association of DNA to the binary complex of Cas9 and guide-RNA is rate-limiting during the first catalytic turnover, while DNA cleavage from a pre-formed ternary complex of Cas9, guide-RNA, and DNA is rapid. Moreover, an extremely slow release of DNA products essentially restricts Cas9 to be a single-turnover enzyme. By simultaneously measuring the contributions of the HNH and RuvC nuclease activities of Cas9 to DNA cleavage, we also uncovered the kinetic basis by which HNH conformationally regulates the RuvC cleavage activity. Together, our results provide crucial kinetic and functional details regarding Cas9 which will inform gene-editing experiments, guide future research to understand off-target DNA cleavage by Cas9, and aid in the continued development of Cas9 as a biotechnological tool.

  12. Efficient CRISPR/Cas9 Genome Editing of Phytoene desaturase in Cassava.

    PubMed

    Odipio, John; Alicai, Titus; Ingelbrecht, Ivan; Nusinow, Dmitri A; Bart, Rebecca; Taylor, Nigel J

    2017-01-01

    CRISPR/Cas9 has become a powerful genome-editing tool for introducing genetic changes into crop species. In order to develop capacity for CRISPR/Cas9 technology in the tropical staple cassava ( Manihot esculenta ), the Phytoene desaturase ( MePDS ) gene was targeted in two cultivars using constructs carrying gRNAs targeting two sequences within MePDS exon 13. After Agrobacterium -mediated delivery of CRISPR/Cas9 reagents into cassava cells, both constructs induced visible albino phenotypes within cotyledon-stage somatic embryos regenerating on selection medium and the plants regenerated therefrom. A total of 58 (cv. 60444) and 25 (cv. TME 204) plant lines were recovered, of which 38 plant lines (19 from each cultivar) were analyzed for mutagenesis. The frequency of plant lines showing albino phenotype was high, ranging from 90 to 100% in cv. TME 204. Observed albino phenotypes were comprised of full albinos devoid of green tissue and chimeras containing a mixture of white and green tissues. Sequence analysis revealed that 38/38 (100%) of the plant lines examined carried mutations at the targeted MePDS site, with insertions, deletions, and substitutions recorded. One putatively mono-allelic homozygous line (1/19) was found from cv. 60444, while 1 (1/19) and 4 (4/19) putatively bi-allelic homozygous lines were found in 60444 and TME204, respectively. The remaining plant lines, comprised mostly of the chimeras, were found to be putatively heterozygous. We observed minor (1 bp) nucleotide substitutions and or deletions upstream of the 5' and or downstream of the 3' targeted MePDS region. The data reported demonstrates that CRISPR/Cas9-mediated genome editing of cassava is highly efficient and relatively simple, generating multi-allelic mutations in both cultivars studied. Modification of MePDS described here generates visually detectable mutated events in a relatively short time frame of 6-8 weeks, and does not require sequencing to confirm editing at the target. It

  13. Closure Report for Corrective Action Unit 415: Project 57 No. 1 Plutonium Dispersion (NTTR) Nevada Test and Training Range, Nevada, Revision 0 with ROTC-1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cabble, Kevin J.; Boehlecke, Robert F.

    This Closure Report (CR) presents information supporting the closure of Corrective Action Unit (CAU) 415: Project 57 No. 1 Plutonium Dispersion, which is located on Range 4808A of the Nevada Test and Training Range (NTTR). This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; U.S. Department of Energy (DOE), Environmental Management; U.S. Department of Defense; and DOE, Legacy Management. CAU 415 comprises one corrective action site (CAS): NAFR-23-02, Pu Contaminated Soil. The purpose of this CR is to provide justification and documentation supporting the recommendationmore » that no further corrective action is needed for CAU 415 based on the implementation of the corrective action of Closure in Place.« less

  14. CRISPR/Cas9: Transcending the Reality of Genome Editing.

    PubMed

    Chira, Sergiu; Gulei, Diana; Hajitou, Amin; Zimta, Alina-Andreea; Cordelier, Pierre; Berindan-Neagoe, Ioana

    2017-06-16

    With the expansion of the microbiology field of research, a new genome editing tool arises from the biology of bacteria that holds the promise of achieving precise modifications in the genome with a simplicity and versatility that surpasses previous genome editing methods. This new technique, commonly named CRISPR/Cas9, led to a rapid expansion of the biomedical field; more specifically, cancer characterization and modeling have benefitted greatly from the genome editing capabilities of CRISPR/Cas9. In this paper, we briefly summarize recent improvements in CRISPR/Cas9 design meant to overcome the limitations that have arisen from the nuclease activity of Cas9 and the influence of this technology in cancer research. In addition, we present challenges that might impede the clinical applicability of CRISPR/Cas9 for cancer therapy and highlight future directions for designing CRISPR/Cas9 delivery systems that might prove useful for cancer therapeutics. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  15. High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells.

    PubMed

    Steyer, Benjamin; Carlson-Stevermer, Jared; Angenent-Mari, Nicolas; Khalil, Andrew; Harkness, Ty; Saha, Krishanu

    2016-04-01

    in the human genome and precisely suture in therapeutic sequences. Biomaterials based delivery strategies could help transition these technologies to the clinic. The design space for materials based delivery strategies is vast and optimization is essential to ensuring the safety and efficacy of these treatments. Therefore, new methods are required to rapidly and systematically screen gene-editing efficacy in human cells. This work utilizes an innovative platform to generate and screen many formulations of synthetic biomaterials and components of the CRISPR-Cas9 system in parallel. On this platform, we watch genome surgery in action using high content image analysis. These capabilities enabled us to identify formulation parameters for Cas9-material complexes that can optimize gene-editing in a specific human cell type. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  16. Interference activity of a minimal Type I CRISPR–Cas system from Shewanella putrefaciens

    PubMed Central

    Dwarakanath, Srivatsa; Brenzinger, Susanne; Gleditzsch, Daniel; Plagens, André; Klingl, Andreas; Thormann, Kai; Randau, Lennart

    2015-01-01

    Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs. PMID:26350210

  17. CRISPR-Cas Technologies and Applications in Food Bacteria.

    PubMed

    Stout, Emily; Klaenhammer, Todd; Barrangou, Rodolphe

    2017-02-28

    Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form adaptive immune systems that occur in many bacteria and most archaea. In addition to protecting bacteria from phages and other invasive mobile genetic elements, CRISPR-Cas molecular machines can be repurposed as tool kits for applications relevant to the food industry. A primary concern of the food industry has long been the proper management of food-related bacteria, with a focus on both enhancing the outcomes of beneficial microorganisms such as starter cultures and probiotics and limiting the presence of detrimental organisms such as pathogens and spoilage microorganisms. This review introduces CRISPR-Cas as a novel set of technologies to manage food bacteria and offers insights into CRISPR-Cas biology. It primarily focuses on the applications of CRISPR-Cas systems and tools in starter cultures and probiotics, encompassing strain-typing, phage resistance, plasmid vaccination, genome editing, and antimicrobial activity.

  18. Diversity and evolution of class 2 CRISPR–Cas systems

    PubMed Central

    Shmakov, Sergey; Smargon, Aaron; Scott, David; Cox, David; Pyzocha, Neena; Yan, Winston; Abudayyeh, Omar O.; Gootenberg, Jonathan S.; Makarova, Kira S.; Wolf, Yuri I.; Severinov, Konstantin; Zhang, Feng; Koonin, Eugene V.

    2018-01-01

    Class 2 CRISPR–Cas systems are characterized by effector modules that consist of a single multidomain protein, such as Cas9 or Cpf1. We designed a computational pipeline for the discovery of novel class 2 variants and used it to identify six new CRISPR–Cas subtypes. The diverse properties of these new systems provide potential for the development of versatile tools for genome editing and regulation. In this Analysis article, we present a comprehensive census of class 2 types and class 2 subtypes in complete and draft bacterial and archaeal genomes, outline evolutionary scenarios for the independent origin of different class 2 CRISPR–Cas systems from mobile genetic elements, and propose an amended classification and nomenclature of CRISPR–Cas. PMID:28111461

  19. Closure Report for Corrective Action Unit 465: Hydronuclear Nevada National Security Site, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mark Burmeister and Patrick Matthews

    2012-11-01

    The corrective action sites (CASs) within CAU 465 are located within Areas 6 and 27 of the NNSS. CAU 465 comprises the following CASs: • 00-23-01, Hydronuclear Experiment, located in Area 27 of the NNSS and known as the Charlie site. • 00-23-02, Hydronuclear Experiment, located in Area 27 of the NNSS and known as the Dog site. • 00-23-03, Hydronuclear Experiment, located in Area 27 of the NNSS and known as the Charlie Prime and Anja sites. • 06-99-01, Hydronuclear, located in Area 6 of the NNSS and known as the Trailer 13 site. The purpose of this CRmore » is to provide documentation supporting the completed corrective actions and provide data confirming that the closure objectives for CASs within CAU 465 were met. From September 2011 through July 2012, closure activities were performed as set forth in the Streamlined Approach for Environmental Restoration Plan for CAU 465: Hydronuclear, Nevada National Security Site, Nevada.« less

  20. Remedial action suitability for the Cornhusker Army Ammunition Plant site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nonavinakere, S.; Rappa, P. III

    1995-12-31

    Numerous Department of Defense (DOD) sites across the nation are contaminated with explosive wastes due to munitions production during World War II, Korean Conflict and Vietnam Conflict. Production activities included explosives manufacturing, loading, packing, assembling, machining, casting and curing. Contaminants often present at these sites include TNT, RDX, HMX, Tetryl 2,4-DNT, 2,6-DNT, 1,3-DNB, 1,3,5-TNB and nitrobenzene. The Cornhusker Army Ammunition Plant (CAAP) is one such DOD site that has been determined to be contaminated with explosives. The CAAP is located approximately 2 miles west of the City of Grand Island in Hall County, Nebraska. The plant produced artillery, bombs, boosters,more » supplementary charges and various other experimental explosives. The purpose of this paper is to provide an overview of the site background, review of the remedial alternatives evaluation process and rationale behind the selection of present remedial action.« less

  1. Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi

    Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing,more » Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.« less

  2. Genome engineering through CRISPR/Cas9 technology in the human germline and pluripotent stem cells.

    PubMed

    Vassena, R; Heindryckx, B; Peco, R; Pennings, G; Raya, A; Sermon, K; Veiga, A

    2016-06-01

    With the recent development of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing technology, the possibility to genetically manipulate the human germline (gametes and embryos) has become a distinct technical possibility. Although many technical challenges still need to be overcome in order to achieve adequate efficiency and precision of the technology in human embryos, the path leading to genome editing has never been simpler, more affordable, and widespread. In this narrative review we seek to understand the possible impact of CRISR/Cas9 technology on human reproduction from the technical and ethical point of view, and suggest a course of action for the scientific community. This non-systematic review was carried out using Medline articles in English, as well as technical documents from the Human Fertilisation and Embryology Authority and reports in the media. The technical possibilities of the CRISPR/Cas9 technology with regard to human reproduction are analysed based on results obtained in model systems such as large animals and laboratory rodents. Further, the possibility of CRISPR/Cas9 use in the context of human reproduction, to modify embryos, germline cells, and pluripotent stem cells is reviewed based on the authors' expert opinion. Finally, the possible uses and consequences of CRISPR/cas9 gene editing in reproduction are analysed from the ethical point of view. We identify critical technical and ethical issues that should deter from employing CRISPR/Cas9 based technologies in human reproduction until they are clarified. Overcoming the numerous technical limitations currently associated with CRISPR/Cas9 mediated editing of the human germline will depend on intensive research that needs to be transparent and widely disseminated. Rather than a call to a generalized moratorium, or banning, of this type of research, efforts should be placed on establishing an open, international, collaborative and regulated research

  3. Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy.

    PubMed

    Nakajima, Osamu; Nishimaki-Mogami, Tomoko; Kondo, Kazunari

    2016-01-01

    Genome editing has undergone rapid development during the last three years. It is anticipated that genetically modified organisms (GMOs) for food purposes will be widely produced using the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR)/Cas9 system in the near future. However, the Cas9 gene may then enter the genomes of GMOs for food if the breeding process is not strictly managed, which could lead to the Cas9 protein or associated peptides being produced within these organisms. A variety of peptides could theoretically be produced from the Cas9 gene by using open reading frames different from that of Cas9 in the GMOs. In this study, Cas9 and the peptides potentially encoded by Cas9 genes were studied regarding their immunogenicity, in terms of the digestibility of Cas9 and the homology of the peptides to food allergens. First, the digestibility and thermal stability of Cas9 were studied. Digestibility was tested with natural or heat-denatured Cas9 in simulated gastric fluid in vitro. The two types of Cas9 were digested rapidly. Cas9 was also gradually degraded during heat treatment. Second, the peptides potentially encoded by Cas9 genes were examined for their homology to food allergens. Specifically, an 8-mer exact match search and a sliding 80-mer window search were performed using allergen databases. One of the peptides was found to have homology with a food allergen.

  4. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... later award of a CAS-covered contract. Full coverage applies to contractor business units that— (1) Receive a single CAS-covered contract award of $50 million or more; or (2) Received $50 million or more in net CAS-covered awards during its preceding cost accounting period. (b) Modified coverage. (1...

  5. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... later award of a CAS-covered contract. Full coverage applies to contractor business units that— (1) Receive a single CAS-covered contract award of $50 million or more; or (2) Received $50 million or more in net CAS-covered awards during its preceding cost accounting period. (b) Modified coverage. (1...

  6. Interference activity of a minimal Type I CRISPR-Cas system from Shewanella putrefaciens.

    PubMed

    Dwarakanath, Srivatsa; Brenzinger, Susanne; Gleditzsch, Daniel; Plagens, André; Klingl, Andreas; Thormann, Kai; Randau, Lennart

    2015-10-15

    Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Exploring the mechanistic insights of Cas scaffolding protein family member 4 with protein tyrosine kinase 2 in Alzheimer's disease by evaluating protein interactions through molecular docking and dynamic simulations.

    PubMed

    Hassan, Mubashir; Shahzadi, Saba; Alashwal, Hany; Zaki, Nazar; Seo, Sung-Yum; Moustafa, Ahmed A

    2018-05-22

    Cas scaffolding protein family member 4 and protein tyrosine kinase 2 are signaling proteins, which are involved in neuritic plaques burden, neurofibrillary tangles, and disruption of synaptic connections in Alzheimer's disease. In the current study, a computational approach was employed to explore the active binding sites of Cas scaffolding protein family member 4 and protein tyrosine kinase 2 proteins and their significant role in the activation of downstream signaling pathways. Sequential and structural analyses were performed on Cas scaffolding protein family member 4 and protein tyrosine kinase 2 to identify their core active binding sites. Molecular docking servers were used to predict the common interacting residues in both Cas scaffolding protein family member 4 and protein tyrosine kinase 2 and their involvement in Alzheimer's disease-mediated pathways. Furthermore, the results from molecular dynamic simulation experiment show the stability of targeted proteins. In addition, the generated root mean square deviations and fluctuations, solvent-accessible surface area, and gyration graphs also depict their backbone stability and compactness, respectively. A better understanding of CAS and their interconnected protein signaling cascade may help provide a treatment for Alzheimer's disease. Further, Cas scaffolding protein family member 4 could be used as a novel target for the treatment of Alzheimer's disease by inhibiting the protein tyrosine kinase 2 pathway.

  8. No Further Action Decision Under CERCLA Study Area 43E Historic Gas Station Sites Fort Devens, Massachusetts

    DTIC Science & Technology

    1995-01-01

    AREA 43E * HISTORIC GAS STATION SITES g FORT DEVENS , MASSACHUSETTS £ I CONTRACT DAAA15-91-D-0008I U.S. ARMY ENVIRONMENTAL CENTER3 ABERDEEN PROVING...FURTHER ACTION DECISION UNDER CERCLA STUDY AREA 43E HISTORIC GAS STATION SITES I FORT DEVENS , MASSACHUSETTS i I 1 Prepared for: U.S. Army...7053-12 JANUARY 1995 I I. NO FURTHER ACTION DECISION UNDER CERCLA STUDY AREA 43E HISTORIC GAS STATION SITES FORT DEVENS , MASSACHUSETTS TABLE OF CONTENTS

  9. Circulating leptin and inflammatory response in esophageal cancer, esophageal cancer-related cachexia-anorexia syndrome (CAS) and non-malignant CAS of the alimentary tract.

    PubMed

    Diakowska, Dorota; Krzystek-Korpacka, Malgorzata; Markocka-Maczka, Krystyna; Diakowski, Witold; Matusiewicz, Malgorzata; Grabowski, Krzysztof

    2010-08-01

    We investigated the association between esophageal cancer and cachexia-anorexia syndrome (CAS) of the alimentary tract and leptin, an adipocytokine crucial for body weight regulation, a modulator of inflammatory/immune response, implication of which in cancer and CAS development remains debatable. Circulating leptin was measured in 135 esophageal cancer patients (51 non-cachectic and 84 cachectic) and 83 controls (63 non-cachectic and 20 cachectic) and referred to cancer stage, CAS, and inflammatory and nutritional indices. Leptin was down-regulated in cancer patients and cachectic controls as compared to non-cachectic controls, with more pronounced hypoleptinemia in advanced cancers. Leptin correlated directly with BMI, TNF-alpha, albumin, and hemoglobin and indirectly with IL-6, IL-8, and hsCRP. The correlations, except for hsCRP, were more pronounced in females. BMI alone (females) and BMI and hsCRP (males) were independent predictors of leptin explaining over 60% of its variability. Following adjustment for BMI and gender, cancer-related CAS but not cancer itself negatively affected leptin. Leptin and BMI were independently associated with cancer-related and non-malignant CAS with diagnostic accuracy of 93% in identifying subjects with CAS. Pro-inflammatory, angiogenic and mitogenic properties of leptin do not seem to be important for esophageal cancer development but hypoleptinemia, independently from co-occurring reduction of adiposity, appears to be strongly associated with esophageal cancer-related CAS and non-malignant CAS of the alimentary tract. Copyright 2010 Elsevier Ltd. All rights reserved.

  10. The CRISPR-Cas system in Enterobacteriaceae.

    PubMed

    Medina-Aparicio, Liliana; Dávila, Sonia; Rebollar-Flores, Javier E; Calva, Edmundo; Hernández-Lucas, Ismael

    2018-02-01

    In nature, microorganisms are constantly exposed to multiple viral infections and thus have developed many strategies to survive phage attack and invasion by foreign DNA. One of such strategies is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) bacterial immunological system. This defense mechanism is widespread in prokaryotes including several families such as Enterobacteriaceae. Much knowledge about the CRISPR-Cas system has been generated, including its biological functions, transcriptional regulation, distribution, utility as a molecular marker and as a tool for specific genome editing. This review focuses on these aspects and describes the state of the art of the CRISPR-Cas system in the Enterobacteriaceae bacterial family. © FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.

    PubMed

    Dehler, Carola E; Boudinot, Pierre; Martin, Samuel A M; Collet, Bertrand

    2016-08-01

    CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.

  12. 78 FR 21352 - Update on Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-10

    ... DEPARTMENT OF ENERGY Update on Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of Energy. ACTION: Notice of the Title X claims during fiscal... at active uranium and thorium processing sites to remediate byproduct material generated as an...

  13. No Further Action Decision Document for Site 8 at Alpena Combat Readiness Training Center, Alpena, Michigan.

    DTIC Science & Technology

    1998-04-01

    INSTALLATION RESTORATION PROGRAM No FURTHER REMEDIAL ACTION PLANNED DECISION DOCUMENT FOR SITE 8 FINAL MICHIGAN AIR NATIONAL GUARD ALPENA ...COMBAT READINESS TRAINING CENTER ALPENA , MICHIGAN April 1998 Air National Guard Andrews AFB, Maryland fr r=.~r i^:r^f>^’ m% Approved for public...Document 4. TITLE AND SUBTITLE No Further Action Decision Document for Site 8 at Alpena CRTC, Alpena , MI. 6. AUTHOR(S) N/A 7. PERFORMING

  14. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation

    PubMed Central

    Doench, John G.; Hartenian, Ella; Graham, Daniel B.; Tothova, Zuzana; Hegde, Mudra; Smith, Ian; Sullender, Meagan; Ebert, Benjamin L.; Xavier, Ramnik J.; Root, David E.

    2014-01-01

    Components of the prokaryotic clustered regularly interspersed palindromic repeat (CRISPR) loci have recently been repurposed for use in mammalian cells1–6. The Cas9 protein can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system7,8. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the proto-spacer adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest. PMID:25184501

  15. Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system.

    PubMed

    Liu, Xiaoxi; Homma, Ayaka; Sayadi, Jamasb; Yang, Shu; Ohashi, Jun; Takumi, Toru

    2016-01-27

    The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications.

  16. Applications of CRISPR/Cas System to Bacterial Metabolic Engineering.

    PubMed

    Cho, Suhyung; Shin, Jongoh; Cho, Byung-Kwan

    2018-04-05

    The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi) system including deactivated Cas9 (dCas9) with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome. This review discusses the applications of CRISPR/Cas for genome editing in various bacterial systems and their applications. In particular, CRISPR technology has been used for the production of metabolites of high industrial significance, including biochemical, biofuel, and pharmaceutical products/precursors in bacteria. Here, we focus on methods to increase the productivity and yield/titer scan by controlling metabolic flux through individual or combinatorial use of CRISPR/Cas and CRISPRi systems with introduction of synthetic pathway in industrially common bacteria including Escherichia coli . Further, we discuss additional useful applications of the CRISPR/Cas system, including its use in functional genomics.

  17. The genome editing revolution: A CRISPR-Cas TALE off-target story.

    PubMed

    Stella, Stefano; Montoya, Guillermo

    2016-07-01

    In the last 10 years, we have witnessed a blooming of targeted genome editing systems and applications. The area was revolutionized by the discovery and characterization of the transcription activator-like effector proteins, which are easier to engineer to target new DNA sequences than the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (clustered regularly interspaced short palindromic sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR-Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human pathways or to improve key organisms for biotechnological applications, such as plants, livestock genome as well as yeasts and bacterial strains. © 2016 The Authors. BioEssays published by WILEY Periodicals, Inc.

  18. Whole genome analysis of CRISPR Cas9 sgRNA off-target homologies via an efficient computational algorithm.

    PubMed

    Zhou, Hong; Zhou, Michael; Li, Daisy; Manthey, Joseph; Lioutikova, Ekaterina; Wang, Hong; Zeng, Xiao

    2017-11-17

    The beauty and power of the genome editing mechanism, CRISPR Cas9 endonuclease system, lies in the fact that it is RNA-programmable such that Cas9 can be guided to any genomic loci complementary to a 20-nt RNA, single guide RNA (sgRNA), to cleave double stranded DNA, allowing the introduction of wanted mutations. Unfortunately, it has been reported repeatedly that the sgRNA can also guide Cas9 to off-target sites where the DNA sequence is homologous to sgRNA. Using human genome and Streptococcus pyogenes Cas9 (SpCas9) as an example, this article mathematically analyzed the probabilities of off-target homologies of sgRNAs and discovered that for large genome size such as human genome, potential off-target homologies are inevitable for sgRNA selection. A highly efficient computationl algorithm was developed for whole genome sgRNA design and off-target homology searches. By means of a dynamically constructed sequence-indexed database and a simplified sequence alignment method, this algorithm achieves very high efficiency while guaranteeing the identification of all existing potential off-target homologies. Via this algorithm, 1,876,775 sgRNAs were designed for the 19,153 human mRNA genes and only two sgRNAs were found to be free of off-target homology. By means of the novel and efficient sgRNA homology search algorithm introduced in this article, genome wide sgRNA design and off-target analysis were conducted and the results confirmed the mathematical analysis that for a sgRNA sequence, it is almost impossible to escape potential off-target homologies. Future innovations on the CRISPR Cas9 gene editing technology need to focus on how to eliminate the Cas9 off-target activity.

  19. Analysis of microsatellite instability in CRISPR/Cas9 editing mice.

    PubMed

    Huo, Xueyun; Du, Yating; Lu, Jing; Guo, Meng; Li, Zhenkun; Zhang, Shuangyue; Li, Xiaohong; Chen, Zhenwen; Du, Xiaoyan

    2017-03-01

    Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated (Cas) protein 9 system is a novel and powerful tool which is widely used for genome editing. CRISPR/Cas9 is RNA-guided and can lead to desired genomic modifications. However, whether the CRISPR/Cas9-mediated genome editing causes genomic alterations and genomic instability, such as microsatellite instability (MSI), is still unknown. Here we detected MSI in 21 CRISPR/Cas9 mouse strains using a panel of 42 microsatellite loci which were selected from our previous studies. Surprisingly, MSI occurrence was common in CRISPR/Cas9 modified genome, and most of the strains (19/21, 90.5%) examined showed MSI. Of 42 loci examined, 8 loci (8/42, 19.05%) exhibited MSI in the Cas9 editing mice. The Ttll9 (4/42, 9.5%) were the most unstable strains, and D10Mit3 and D10Mit198 (9/21, 42.9%) were considered to be the most "hot" loci in the Cas9 strains we tested. Through analyzing the mutation of microsatellite loci, we provide new insights into the genomic alterations of CRISPR/Cas9 models and it will help us for a better understanding of this powerful technology. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Environmental assessment of remedial action at the Gunnison Uranium Mill Tailings Site, Gunnison, Colorado. [UMTRA Project

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bachrach, A.; Hoopes, J.; Morycz, D.

    1984-12-01

    This document assesses and compares the environmental impacts of various alternatives for remedial action at the Gunnison uranium of mill tailings site located 0.5 miles south of Gunnison, Colorado. The site covers 56 acres and contains 35 acres of tailings, 2 of the original mill buildings and a water tower. The Uranium Mill Tailings Radiation Control of Act of 1978 (UMTRCA), Public Law 95-604, authorizes the US Department of Energy to clean up the site to reduce the potential health impacts associated with the residual radioactive materials remaining at the site and at associated (vicinity) properties off the site. Themore » US Environmental Protection Agency promulgated standards for the remedial actions (40 CFR 192). Remedial actions must be performed in accordance with these standards and with the occurrence of the Nuclear Regulatory Commission. Four alternatives have been addressed in this document. The first alternative is to consolidate the tailings and associated contaminated soils into a recontoured pile on the southern portion of the existing site. A radon barrier of silty clay would be constructed over the pile and various erosion control measures would be taken to assure the long-term integrity of the pile. Two other alternatives which involve moving the tailings to new locations are assessed in this document. These alternatives generally involve greater short-term impacts and are more costly but would result in the tailings being stabilized in a location farther from the city of Gunnison. The no action alternative is also assessed.« less

  1. Environmental Assessment of remedial action at the Ambrosia Lake uranium mill tailings site, Ambrosia Lake, New Mexico

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Not Available

    1987-06-01

    This document assesses and compares the environmental impacts of various alternatives for remedial action at the Ambrosia Lake uranium mill tailings site located near Ambrosia Lake, New Mexico. The designated site covers 196 acres and contains 111 acres of tailings and some of the original mill structures. The Uranium Mill Tailings Radiation Control Act (UMTRCA), Public Law 95-604, authorizes the US Department of Energy to clean up the site to reduce the potential health impacts associated with the residual radioactive materials remaining at the site and at associated properties off the site. The US Environmental Protection Agency promulgated standards formore » th remedial action (40 CFR Part 192). Remedial action must be performed in accordance with these standards and with the concurrence of the Nuclear Regulatory Commission. The proposed action is to stabilize the tailings at their present location by consolidating the tailings and associated contaminated materials into a recontoured pile. A radon barrier would be constructed over the pile and various erosion protection measures would be taken to assure the long-term stability of the pile. Another alternative which would involve moving the tailings to a new location is also assessed in this document. This alternative would generally involve greater short-term impacts and costs but would result in stabilization of the tailings at an undeveloped location. The no action alternative is also assessed in this document.« less

  2. CAS as Environments for Implementing Mathematical Microworlds.

    ERIC Educational Resources Information Center

    Alpers, Burkhard

    2002-01-01

    Investigates whether computer algebra systems (CAS) are suitable environments for implementing mathematical microworlds. Recalls what constitutes a microworld and explores how CAS can be used for implementation, stating potentials as well as limitations. Provides as an example the microworld "Formula 1", implemented in Maple Software. (Author/KHR)

  3. Two Distinct Approaches for CRISPR-Cas9-Mediated Gene Editing in Cryptococcus neoformans and Related Species.

    PubMed

    Wang, Ping

    2018-06-27

    Cryptococcus neoformans and related species are encapsulated basidiomycetous fungi that cause meningoencephalitis in individuals with immune deficiency. This pathogen has a tractable genetic system; however, gene disruption via electroporation remains difficult, while biolistic transformation is often limited by lack of multiple genetic markers and the high initial cost of equipment. The approach using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) has become the technology of choice for gene editing in many organisms due to its simplicity, efficiency, and versatility. The technique has been successfully demonstrated in C. neoformans and Cryptococcus deneoformans in which two DNA plasmids expressing either the Streptococcus pyogenes CAS9 gene or the guide RNA (gRNA) were employed. However, potential adverse effects due to constitutive expression and the time-consuming process of constructing vectors to express each gRNA remain as a primary barrier for wide adaptation. This report describes the delivery of preassembled CRISPR-Cas9-gRNA ribonucleoproteins (RNPs) via electroporation that is able to generate edited mutant alleles. RNP-mediated CRISPR-Cas9 was used to replace the wild-type GIB2 gene encoding a Gβ-like/RACK1 Gib2 protein with a gib2 :: NAT allele via homologous recombination in both C. neoformans and C. deneoformans In addition, a DNA plasmid (pCnCas9:U6-gRNA) that expresses both Cas9 and gRNA, allowing for convenient yet low-cost DNA-mediated gene editing, is described. pCnCas9:U6-gRNA contains an endogenous U6 promoter for gRNA expression and restriction sites for one-step insertion of a gRNA. These approaches and resources provide new opportunities to accelerate genetic studies of Cryptococcus species. IMPORTANCE For genetic studies of the Cryptococcus genus, generation of mutant strains is often hampered by a limited number of selectable genetic markers, the tedious process of vector

  4. From Bioengineering to CRISPR/Cas9 – A Personal Retrospective of 20 Years of Research in Programmable Genome Targeting

    PubMed Central

    Jeltsch, Albert

    2018-01-01

    Genome targeting of restriction enzymes and DNA methyltransferases has many important applications including genome and epigenome editing. 15–20 years ago, my group was involved in the development of approaches for programmable genome targeting, aiming to connect enzymes with an oligodeoxynucleotide (ODN), which could form a sequence-specific triple helix at the genomic target site. Importantly, the target site of such enzyme-ODN conjugate could be varied simply by altering the ODN sequence promising great applicative values. However, this approach was facing many problems including the preparation and purification of the enzyme-ODN conjugates, their efficient delivery into cells, slow kinetics of triple helix formation and the requirement of a poly-purine target site sequence. Hence, for several years genome and epigenome editing approaches mainly were based on Zinc fingers and TAL proteins as targeting devices. More recently, CRISPR/Cas systems were discovered, which use a bound RNA for genome targeting that forms an RNA/DNA duplex with one DNA strand of the target site. These systems combine all potential advantages of the once imagined enzyme-ODN conjugates and avoid all main disadvantageous. Consequently, the application of CRISPR/Cas in genome and epigenome editing has exploded in recent years. We can draw two important conclusions from this example of research history. First, evolution still is the better bioengineer than humans and, whenever tested in parallel, natural solutions outcompete engineered ones. Second, CRISPR/Cas system were discovered in pure, curiosity driven, basic research, highlighting that it is basic, bottom-up research paving the way for fundamental innovation. PMID:29434619

  5. Single step production of Cas9 mRNA for zygote injection.

    PubMed

    Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

    2018-03-01

    Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

  6. Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response.

    PubMed

    Heler, Robert; Wright, Addison V; Vucelja, Marija; Bikard, David; Doudna, Jennifer A; Marraffini, Luciano A

    2017-01-05

    CRISPR loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the invading DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. We identified a mutation, I473F, that increases the rate of spacer acquisition by more than two orders of magnitude. Our results highlight the role of Cas9 during CRISPR immunization and provide a useful tool to study this rare process and develop it as a biotechnological application. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Survey of clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) systems in multiple sequenced strains of Klebsiella pneumoniae.

    PubMed

    Ostria-Hernández, Martha Lorena; Sánchez-Vallejo, Carlos Javier; Ibarra, J Antonio; Castro-Escarpulli, Graciela

    2015-08-04

    In recent years the emergence of multidrug resistant Klebsiella pneumoniae strains has been an increasingly common event. This opportunistic species is one of the five main bacterial pathogens that cause hospital infections worldwide and multidrug resistance has been associated with the presence of high molecular weight plasmids. Plasmids are generally acquired through horizontal transfer and therefore is possible that systems that prevent the entry of foreign genetic material are inactive or absent. One of these systems is CRISPR/Cas. However, little is known regarding the clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) system in K. pneumoniae. The adaptive immune system CRISPR/Cas has been shown to limit the entry of foreign genetic elements into bacterial organisms and in some bacteria it has been shown to be involved in regulation of virulence genes. Thus in this work we used bioinformatics tools to determine the presence or absence of CRISPR/Cas systems in available K. pneumoniae genomes. The complete CRISPR/Cas system was identified in two out of the eight complete K. pneumoniae genomes sequences and in four out of the 44 available draft genomes sequences. The cas genes in these strains comprises eight cas genes similar to those found in Escherichia coli, suggesting they belong to the type I-E group, although their arrangement is slightly different. As for the CRISPR sequences, the average lengths of the direct repeats and spacers were 29 and 33 bp, respectively. BLAST searches demonstrated that 38 of the 116 spacer sequences (33%) are significantly similar to either plasmid, phage or genome sequences, while the remaining 78 sequences (67%) showed no significant similarity to other sequences. The region where the CRISPR/Cas systems were located is the same in all the Klebsiella genomes containing it, it has a syntenic architecture, and is located among genes encoding for proteins likely involved in

  8. Fanconi anemia gene editing by the CRISPR/Cas9 system.

    PubMed

    Osborn, Mark J; Gabriel, Richard; Webber, Beau R; DeFeo, Anthony P; McElroy, Amber N; Jarjour, Jordan; Starker, Colby G; Wagner, John E; Joung, J Keith; Voytas, Daniel F; von Kalle, Christof; Schmidt, Manfred; Blazar, Bruce R; Tolar, Jakub

    2015-02-01

    Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, because of its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder.

  9. Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens.

    PubMed

    Morgens, David W; Wainberg, Michael; Boyle, Evan A; Ursu, Oana; Araya, Carlos L; Tsui, C Kimberly; Haney, Michael S; Hess, Gaelen T; Han, Kyuho; Jeng, Edwin E; Li, Amy; Snyder, Michael P; Greenleaf, William J; Kundaje, Anshul; Bassik, Michael C

    2017-05-05

    CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.

  10. Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens

    PubMed Central

    Morgens, David W.; Wainberg, Michael; Boyle, Evan A.; Ursu, Oana; Araya, Carlos L.; Tsui, C. Kimberly; Haney, Michael S.; Hess, Gaelen T.; Han, Kyuho; Jeng, Edwin E.; Li, Amy; Snyder, Michael P.; Greenleaf, William J.; Kundaje, Anshul; Bassik, Michael C.

    2017-01-01

    CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens. PMID:28474669

  11. A non-inheritable maternal Cas9-based multiple-gene editing system in mice.

    PubMed

    Sakurai, Takayuki; Kamiyoshi, Akiko; Kawate, Hisaka; Mori, Chie; Watanabe, Satoshi; Tanaka, Megumu; Uetake, Ryuichi; Sato, Masahiro; Shindo, Takayuki

    2016-01-28

    The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9 overexpression (Cas9 mice). The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome. The efficiency of such maCas9-based genome editing was comparable to that of zygote microinjection-based genome editing widely used at present. Furthermore, we demonstrated a novel approach to create "Cas9 transgene-free" gene-modified mice using non-Tg (+/+) zygotes carrying maCas9. The maCas9 protein in mouse zygotes edited nine target loci simultaneously after injection with nine different gRNAs alone. Cas9 mouse-derived zygotes have the potential to facilitate the creation of genetically modified animals carrying the Cas9 transgene, enabling repeatable genome engineering and the production of Cas9 transgene-free mice.

  12. Addendum to the Corrective Action Decision Document/Closure Report for Corrective Action Unit 406: Area 3 Building 03-74 & Building 03-58 Underground Discharge Points and Corrective Action Unit 429: Area 3 Building 03-55 & Area 9 Building 09-52 Underground Discharge Points, Tonopah Test Range, Nevada, Revision 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lynn Kidman

    This document constitutes an addendum to the March 2000, Corrective Action Decision Document / Closure Report for Corrective Action Unit 406: Area 3 Building 03-74 & 03-58 Underground Discharge Points and Corrective Action Unit 429: Area 3 Building 03-55 & Area 9 Building 09-52 Underground Discharge Points (TTR) as described in the document Recommendations and Justifications for Modifications for Use Restrictions Established under the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office Federal Facility Agreement and Consent Order (UR Modification document) dated February 2008. The UR Modification document was approved by NDEP on February 26, 2008. Themore » approval of the UR Modification document constituted approval of each of the recommended UR modifications. In conformance with the UR Modification document, this addendum consists of: • This cover page that refers the reader to the UR Modification document for additional information • The cover and signature pages of the UR Modification document • The NDEP approval letter • The corresponding section of the UR Modification document This addendum provides the documentation justifying the cancellation of the UR for CAS 03-51-001-0355 – Photo Shop UDP, Drains in CAU 429. It should be noted that there are no changes to CAU 406. This UR was established as part of a Federal Facility Agreement and Consent Order (FFACO) corrective action and is based on the presence of contaminants at concentrations greater than the action levels established at the time of the initial investigation (FFACO, 1996; as amended August 2006). Since this UR was established, practices and procedures relating to the implementation of risk-based corrective actions (RBCA) have changed. Therefore, this UR was re-evaluated against the current RBCA criteria as defined in the Industrial Sites Project Establishment of Final Action Levels (NNSA/NSO, 2006c). This re-evaluation consisted of comparing the

  13. Corrective Action Decision Document/Corrective Action Plan for the 92-Acre Area and Corrective Action Unit 111: Area 5 WMD Retired Mixed Waste Pits, Nevada Test Site, Nevada

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NSTec Environmental Restoration

    2009-07-31

    This Corrective Action Decision Document/Corrective Action Plan (CADD/CAP) has been prepared for the 92-Acre Area, the southeast quadrant of the Radioactive Waste Management Site, located in Area 5 of the Nevada Test Site (NTS). The 92-Acre Area includes Corrective Action Unit (CAU) 111, 'Area 5 WMD Retired Mixed Waste Pits.' Data Quality Objectives (DQOs) were developed for the 92-Acre Area, which includes CAU 111. The result of the DQO process was that the 92-Acre Area is sufficiently characterized to provide the input data necessary to evaluate corrective action alternatives (CAAs) without the collection of additional data. The DQOs are includedmore » as Appendix A of this document. This CADD/CAP identifies and provides the rationale for the recommended CAA for the 92-Acre Area, provides the plan for implementing the CAA, and details the post-closure plan. When approved, this CADD/CAP will supersede the existing Pit 3 (P03) Closure Plan, which was developed in accordance with Title 40 Code of Federal Regulations (CFR) Part 265, 'Interim Status Standards for Owners and Operators of Hazardous Waste Treatment, Storage, and Disposal Facilities.' This document will also serve as the Closure Plan and the Post-Closure Plan, which are required by 40 CFR 265, for the 92-Acre Area. After closure activities are complete, a request for the modification of the Resource Conservation and Recovery Act Permit that governs waste management activities at the NTS will be submitted to the Nevada Division of Environmental Protection to incorporate the requirements for post-closure monitoring. Four CAAs, ranging from No Further Action to Clean Closure, were evaluated for the 92-Acre Area. The CAAs were evaluated on technical merit focusing on performance, reliability, feasibility, safety, and cost. Based on the evaluation of the data used to develop the conceptual site model; a review of past, current, and future operations at the site; and the detailed and comparative analysis of

  14. VARIABLE BOUND-SITE CHARGING CONTRIBUTIONS TO SURFACE COMPLEXATION MASS ACTION EXPRESSIONS

    EPA Science Inventory

    One and two pK models of surface complexation reactions between reactive surface sites (>SOH) and the proton (H+) use mass action expressions of the form: Ka={[>SOHn-1z-1]g>SOH(0-1)aH+EXP(-xeY/kT)}/{[>SOHnz]g>SOH(n)} where Ka=the acidity constant, [ ]=reactive species concentrati...

  15. An Introduction to the Tibet cGPS pilot project: TigiCAS

    NASA Astrophysics Data System (ADS)

    Zhang, Z.; Liu, J.; Galetzka, J.; Avouac, J.; Tapponnier, P.; Zeng, L.; Gan, W.; Shen, Z.; Wang, M.

    2007-12-01

    The convergence between India and Eurasia is the¡¡prototype of continental collision in action. Compared¡¡to geological history and fault kinematics studies, the present-day, regional pattern of strain-partitioning¡¡is still inadequately known. Among limited geodetic¡¡efforts in the past decade or two, most have been focused¡¡on refining measurements of the current crustal¡¡shortening rate across the Himalaya. The vast region¡¡immediately to the north is sparsely instrumented, with only one continuous GPS station (Lhasa) within¡¡the plateau proper. Campaign stations are few and¡¡ill-positioned, mostly along major roads, providing¡¡poor constraints on present-day slip-rates on individual¡¡active faults. The extant GPS network configuration is thus still insufficient to discriminate between block vs continuum deformation. In November 2006, the¡¡Chinese Academy of Sciences led a pilot program and¡¡installed 6 continuous GPS stations in southern Tibet, crossing the NS-trending normal fault systems and¡¡complementing the Nepal cGPS profiles. We present¡¡here the new sites, preliminary data processing results, and the spatial relationship with ongoing or planned¡¡continuous GPS sites from a couple of other projects. Together with such projects, TigiCAS will provide¡¡a substantial increase in geodetic data in the¡¡Himalayan-Tibet convergent belt in the next few¡¡years, and lead to a better understanding of¡¡contemporary deformation of the region.

  16. Cas9 gRNA engineering for genome editing, activation and repression

    DOE PAGES

    Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; ...

    2015-09-07

    Here we demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

  17. Generation and validation of PAX7 reporter lines from human iPS cells using CRISPR/Cas9 technology.

    PubMed

    Wu, Jianbo; Hunt, Samuel D; Xue, Haipeng; Liu, Ying; Darabi, Radbod

    2016-03-01

    Directed differentiation of iPS cells toward various tissue progenitors has been the focus of recent research. Therefore, generation of tissue-specific reporter iPS cell lines provides better understanding of developmental stages in iPS cells. This technical report describes an efficient strategy for generation and validation of knock-in reporter lines in human iPS cells using the Cas9-nickase system. Here, we have generated a knock-in human iPS cell line for the early myogenic lineage specification gene of PAX7. By introduction of site-specific double-stranded breaks (DSB) in the genomic locus of PAX7 using CRISPR/Cas9 nickase pairs, a 2A-GFP reporter with selection markers has been incorporated before the stop codon of the PAX7 gene at the last exon. After positive and negative selection, single cell-derived human iPS clones have been isolated and sequenced for in-frame positioning of the reporter construct. Finally, by using a nuclease-dead Cas9 activator (dCas9-VP160) system, the promoter region of PAX7 has been targeted for transient gene induction to validate the GFP reporter activity. This was confirmed by flow cytometry analysis and immunostaining for PAX7 and GFP. This technical report provides a practical guideline for generation and validation of knock-in reporters using CRISPR/Cas9 system. Published by Elsevier B.V.

  18. Nuclear facility decommissioning and site remedial actions: A selected bibliography, Volume 18. Part 1B: Citations with abstracts, sections 10 through 16

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    1997-09-01

    This bibliography contains 3,638 citations with abstracts of documents relevant to environmental restoration, nuclear facility decontamination and decommissioning (D and D), uranium mill tailings management, and site remedial actions. The bibliography contains scientific, technical, financial, and regulatory information that pertains to DOE environmental restoration programs. The citations are separated by topic into 16 sections, including (1) DOE Environmental Restoration Program; (2) DOE D and D Program; (3) Nuclear Facilities Decommissioning; (4) DOE Formerly Utilized sites Remedial Action Program; (5) NORM-Contaminated Site Restoration; (6) DOE Uranium Mill Tailings Remedial Action Project; (7) Uranium Mill Tailings Management; (8) DOE Site-Wide Remedial Actions;more » (9) DOE Onsite Remedial Action Projects; (10) Contaminated Site Remedial Actions; (11) DOE Underground Storage Tank Remediation; (12) DOE Technology Development, Demonstration, and Evaluation; (13) Soil Remediation; (14) Groundwater Remediation; (15) Environmental Measurements, Analysis, and Decision-Making; and (16) Environmental Management Issues.« less

  19. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system.

    PubMed

    Belhaj, Khaoula; Chaparro-Garcia, Angela; Kamoun, Sophien; Nekrasov, Vladimir

    2013-10-11

    Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants.

  20. Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease.

    PubMed

    Yiu, Glenn; Tieu, Eric; Nguyen, Anthony T; Wong, Brittany; Smit-McBride, Zeljka

    2016-10-01

    To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells. CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay. Five gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% ± 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected. The CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.

  1. Mr.CAS-A minimalistic (pure) Ruby CAS for fast prototyping and code generation

    NASA Astrophysics Data System (ADS)

    Ragni, Matteo

    There are Computer Algebra System (CAS) systems on the market with complete solutions for manipulation of analytical models. But exporting a model that implements specific algorithms on specific platforms, for target languages or for particular numerical library, is often a rigid procedure that requires manual post-processing. This work presents a Ruby library that exposes core CAS capabilities, i.e. simplification, substitution, evaluation, etc. The library aims at programmers that need to rapidly prototype and generate numerical code for different target languages, while keeping separated mathematical expression from the code generation rules, where best practices for numerical conditioning are implemented. The library is written in pure Ruby language and is compatible with most Ruby interpreters.

  2. Les tuberculomes intracraniens: à propos de 125 cas

    PubMed Central

    Moufid, Faycal; Oulali, Noureddine; El Fatemi, Nizare; Gana, Rachid; Maaqili, Rachid; Bellakhdar, Fouad

    2012-01-01

    Les tuberculomes intracrâniens représentent l'une des localisations les plus graves de la tuberculose, leur incidence varie en fonction du contexte représentant 0,2% des processus intracrâniens dans les pays occidentaux et 5 à 10% des masses intracrâniennes dans les pays en voie de développement. Nous rapportons une étude rétrospective de 125 cas. L'hypertension intracrânienne (45%) et le déficit neurologique (36%) sont les signes cliniques les plus fréquents. La lésion était localisée dans 60% des cas en sus-tentoriel et dans 40% des cas en sous-tentoriel. L'approche thérapeutique a consisté en un abord direct du tuberculome dans 67 cas (53%), une biopsie stéréotaxique dans 32 cas (25%), le traitement médical en première intention sans confirmation histologique dans 26 cas (20%). Avant 1993 notre service ne disposait pas de cadre de stéréotaxie, notre attitude thérapeutique consistait soit en un abord direct de la lésion dans 70% des cas, soit un traitement antituberculeux en première intention sans confirmation histologique (30%). Cette attitude était corrélée à une mortalité et morbidité non négligeables respectivement 3% et 10%. Après 1993; le taux d'abords direct a chuté a 38%, avec 47% de biopsies stéréotaxiques et seulement 13% des patients traités par antibacillaires sans preuve histologique. Ceci s'est accompagné d'une réduction significative de mortalité a 1,4% (p = 0,0003) et de morbidité a 2% (p = 0,0027). PMID:22937196

  3. Corrective Action Investigation Plan for Corrective Action Unit 516: Septic Systems and Discharge Points, Nevada Test Site, Nevada, Rev. 0, Including Record of Technical Change No. 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    None

    This Corrective Action Investigation Plan (CAIP) contains the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Sites Office's (NNSA/NSO's) approach to collect the data necessary to evaluate corrective action alternatives appropriate for the closure of Corrective Action Unit (CAU) 516, Septic Systems and Discharge Points, Nevada Test Site (NTS), Nevada, under the Federal Facility Agreement and Consent Order. CAU 516 consists of six Corrective Action Sites: 03-59-01, Building 3C-36 Septic System; 03-59-02, Building 3C-45 Septic System; 06-51-01, Sump Piping, 06-51-02, Clay Pipe and Debris; 06-51-03, Clean Out Box and Piping; and 22-19-04, Vehicle Decontamination Area. Located in Areasmore » 3, 6, and 22 of the NTS, CAU 516 is being investigated because disposed waste may be present without appropriate controls, and hazardous and/or radioactive constituents may be present or migrating at concentrations and locations that could potentially pose a threat to human health and the environment. Existing information and process knowledge on the expected nature and extent of contamination of CAU 516 are insufficient to select preferred corrective action alternatives; therefore, additional information will be obtained by conducting a corrective action investigation. The results of this field investigation will support a defensible evaluation of corrective action alternatives in the corrective action decision document. Record of Technical Change No. 1 is dated 3/2004.« less

  4. Cas9-mediated targeting of viral RNA in eukaryotic cells.

    PubMed

    Price, Aryn A; Sampson, Timothy R; Ratner, Hannah K; Grakoui, Arash; Weiss, David S

    2015-05-12

    Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems are prokaryotic RNA-directed endonuclease machineries that act as an adaptive immune system against foreign genetic elements. Using small CRISPR RNAs that provide specificity, Cas proteins recognize and degrade nucleic acids. Our previous work demonstrated that the Cas9 endonuclease from Francisella novicida (FnCas9) is capable of targeting endogenous bacterial RNA. Here, we show that FnCas9 can be directed by an engineered RNA-targeting guide RNA to target and inhibit a human +ssRNA virus, hepatitis C virus, within eukaryotic cells. This work reveals a versatile and portable RNA-targeting system that can effectively function in eukaryotic cells and be programmed as an antiviral defense.

  5. Cas9-mediated targeting of viral RNA in eukaryotic cells

    PubMed Central

    Price, Aryn A.; Sampson, Timothy R.; Ratner, Hannah K.; Grakoui, Arash; Weiss, David S.

    2015-01-01

    Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems are prokaryotic RNA-directed endonuclease machineries that act as an adaptive immune system against foreign genetic elements. Using small CRISPR RNAs that provide specificity, Cas proteins recognize and degrade nucleic acids. Our previous work demonstrated that the Cas9 endonuclease from Francisella novicida (FnCas9) is capable of targeting endogenous bacterial RNA. Here, we show that FnCas9 can be directed by an engineered RNA-targeting guide RNA to target and inhibit a human +ssRNA virus, hepatitis C virus, within eukaryotic cells. This work reveals a versatile and portable RNA-targeting system that can effectively function in eukaryotic cells and be programmed as an antiviral defense. PMID:25918406

  6. Site action, environmental justice and an urban community: A unique approach at a Superfund site

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Seppi, P.K.; Richman, L.R.; Wireman, J.M.

    1994-12-31

    The US Environmental Protection Agency`s (EPA) project at the Diamond Alkali Superfund Site is an example of how technical, environmental justice, and community relations issues all affect actions at a Superfund Site. The Diamond Alkali Superfund Site is divided into two operable units. The site consists of the former pesticides manufacturing facility at 80 and 120 Lister Avenue in Newark, New Jersey, and the adjoining six mile reach of the Passaic River known as the ``Passaic River Study Area``. EPA has negotiated Consent Orders with the Potentially Responsible Party (PRP) to design and construct the selected containment remedy at themore » land-based properties, and to conduct the Remedial Investigation (RI) of the river under EPA oversight. Pesticides, dioxin, polychlorinated biphenyls (PCBs), metals and other hazardous substances have been found at the Site. Evidence indicates that the ecology of the Passaic River has been adversely impacted by the presence of these hazardous substances. The State of New Jersey issued a ban on the consumption of fish and crabs from affected sections of the Passaic River; yet reportedly, many residents still consume seafood from the river. Community relations at the Site had deteriorated because of the community`s lack of trust and loss of confidence in EPA. To address these issues, EPA has implemented an innovative public outreach program to improve how it communicates with racial minority and low-income communities living in the vicinity of the Site, and to involve them in the decision-making process.« less

  7. Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System.

    PubMed

    Nandal, Anjali; Mallon, Barbara; Telugu, Bhanu P

    2017-11-08

    Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide "NGG" protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

  8. An updated evolutionary classification of CRISPR–Cas systems

    PubMed Central

    Makarova, Kira S.; Wolf, Yuri I.; Alkhnbashi, Omer S.; Costa, Fabrizio; Shah, Shiraz A.; Saunders, Sita J.; Barrangou, Rodolphe; Brouns, Stan J. J.; Charpentier, Emmanuelle; Haft, Daniel H.; Horvath, Philippe; Moineau, Sylvain; Mojica, Francisco J. M.; Terns, Rebecca M.; Terns, Michael P.; White, Malcolm F.; Yakunin, Alexander F.; Garrett, Roger A.; van der Oost, John; Backofen, Rolf; Koonin, Eugene V.

    2017-01-01

    The evolution of CRISPR–cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR–cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR–Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized. PMID:26411297

  9. Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System.

    PubMed

    Li, Shenglan; Xue, Haipeng; Wu, Jianbo; Rao, Mahendra S; Kim, Dong H; Deng, Wenbin; Liu, Ying

    2015-12-15

    Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor. Gene targeting efficiency was greatly improved in CRISPR/Cas9-mediated homology directed recombination (∼ 33% correctly targeted clones) compared to conventional targeting protocol (∼ 3%) at the same locus. No off-target events were detected. In addition, taking the advantage of the versatile applications of the CRISPR/Cas9 system, we designed transactivation components to transiently induce NEUROG2 expression, which helps identify transcription factor binding sites and trans-regulation regions of human NEUROG2. The strategy of using CRISPR/Cas9 genome editing coupled with fluorescence-activated cell sorting of neural progenitor cells in a knockin lineage hiPSC reporter platform might be broadly applicable in other stem cell derivatives and subpopulations.

  10. Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System

    PubMed Central

    Li, Shenglan; Xue, Haipeng; Wu, Jianbo; Rao, Mahendra S.; Kim, Dong H.; Deng, Wenbin

    2015-01-01

    Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor. Gene targeting efficiency was greatly improved in CRISPR/Cas9-mediated homology directed recombination (∼33% correctly targeted clones) compared to conventional targeting protocol (∼3%) at the same locus. No off-target events were detected. In addition, taking the advantage of the versatile applications of the CRISPR/Cas9 system, we designed transactivation components to transiently induce NEUROG2 expression, which helps identify transcription factor binding sites and trans-regulation regions of human NEUROG2. The strategy of using CRISPR/Cas9 genome editing coupled with fluorescence-activated cell sorting of neural progenitor cells in a knockin lineage hiPSC reporter platform might be broadly applicable in other stem cell derivatives and subpopulations. PMID:26414932

  11. Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR.

    PubMed

    Kim, Yea Woon; Kim, AeRi

    2017-07-20

    Transcription factors play roles in gene transcription through direct binding to their motifs in genome, and inhibiting this binding provides an effective strategy for studying their roles. Here we applied the CRISPR/spCas9 system to mutate the binding motifs of transcription factors. Binding motifs for erythroid specific transcription factors were mutated in the locus control region hypersensitive sites of the human β-globin locus. Guide RNAs targeting binding motifs were cloned into lentiviral CRISPR vector containing the spCas9 gene, and transduced into MEL/ch11 cells carrying a human chromosome 11. DNA mutations in clonal cells were initially screened by quantitative PCR in genomic DNA and then clarified by sequencing. Mutations in binding motifs reduced occupancy by transcription factors in a chromatin environment. Characterization of mutations revealed that the CRISPR/spCas9 system mainly induced deletions in short regions of <20 bp and preferentially deleted nucleotides around the fifth nucleotide upstream of Protospacer adjacent motifs. These results indicate that the CRISPR/Cas9 system is suitable for mutating the binding motifs of transcription factors, and, consequently, would contribute to elucidate the direct roles of transcription factors. ©2017 The Author(s).

  12. Cornerstones of CRISPR-Cas in drug development and therapy

    PubMed Central

    Fellmann, Christof; Gowen, Benjamin G.; Lin, Pei-Chun; Doudna, Jennifer A.; Corn, Jacob E.

    2017-01-01

    The recent development of CRISPR-Cas systems as easily accessible and programmable tools for genome editing and regulation is spurring a revolution in biology. Paired with the rapid expansion of personalized and reference genomic sequence information, technologies based on CRISPR-Cas are enabling nearly unlimited genetic manipulation even in previously difficult contexts, including human cells. Although much attention has focused on the potential of CRISPR-Cas to cure Mendelian diseases, the technology also holds promise to transform the development of therapies to treat complex heritable and somatic disorders. Here we discuss how CRISPR-Cas can impact the next generation of drugs through accelerating the identification and validation of high-value targets, uncovering high confidence biomarkers and developing differentiated breakthrough therapies. We focus on the promises, pitfalls and hurdles of this revolutionary gene editing technology, and also discuss key aspects of different CRISPR-Cas screening platforms and offer our perspectives on the best practices in genome engineering. PMID:28008168

  13. Temperature effect on CRISPR-Cas9 mediated genome editing.

    PubMed

    Xiang, Guanghai; Zhang, Xingying; An, Chenrui; Cheng, Chen; Wang, Haoyi

    2017-04-20

    Zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) are the most commonly used genome editing tools. Previous studies demonstrated that hypothermia treatment increased the mutation rates induced by ZFNs and TALENs in mammalian cells. Here, we characterize the effect of different culture temperatures on CRISPR-Cas9 mediated genome editing and find that the genome editing efficiency of CRISPR-Cas9 is significantly hampered by hypothermia treatment, unlike ZFN and TALEN. In addition, hyperthermia culture condition enhances genome editing by CRISPR-Cas9 in some cell lines, due to the higher enzyme activity and sgRNA expression level at higher temperature. Our study has implications on CRISPR-Cas9 applications in a broad spectrum of species, many of which do not live at 37°C. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  14. Both TALENs and CRISPR/Cas9 directly target the HBB IVS2-654 (C > T) mutation in β-thalassemia-derived iPSCs.

    PubMed

    Xu, Peng; Tong, Ying; Liu, Xiu-zhen; Wang, Ting-ting; Cheng, Li; Wang, Bo-yu; Lv, Xiang; Huang, Yue; Liu, De-pei

    2015-07-09

    β-Thalassemia is one of the most common genetic blood diseases and is caused by either point mutations or deletions in the β-globin (HBB) gene. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations may be a potential therapeutic strategy for this disease. Due to the low efficiency of typical homologous recombination, endonucleases, including TALENs and CRISPR/Cas9, have been widely used to enhance the gene correction efficiency in patient-derived iPSCs. Here, we designed TALENs and CRISPR/Cas9 to directly target the intron2 mutation site IVS2-654 in the globin gene. We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor. In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs. Finally, TALENs-corrected iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system and detected relatively higher transcription of HBB than the uncorrected cells. This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases.

  15. Cas9, Cpf1 and C2c1/2/3-What's next?

    PubMed

    Nakade, Shota; Yamamoto, Takashi; Sakuma, Tetsushi

    2017-05-04

    Since the rapid emergence of clustered regulatory interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system, developed as a genome engineering tool in 2012-2013, most researchers in the life science field have had a fixated interest in this fascinating technology. CRISPR-Cas9 is an RNA-guided DNA endonuclease system, which consists of Cas9 nuclease defining a few targeting base via protospacer adjacent motif complexed with easily customizable single guide RNA targeting around 20-bp genomic sequence. Although Streptococcus pyogenes Cas9 (SpCas9), one of the Cas9 proteins that applications in genome engineering were first demonstrated, still has wide usage because of its high nuclease activity and broad targeting range, there are several limitations such as large molecular weight and potential off-target effect. In this commentary, we describe various improvements and alternatives of CRISPR-Cas systems, including engineered Cas9 variants, Cas9 homologs, and novel Cas proteins other than Cas9. These variations enable flexible genome engineering with high efficiency and specificity, orthogonal genetic control at multiple gene loci, gene knockdown, or fluorescence imaging of transcripts mediated by RNA targeting, and beyond.

  16. Application of the gene editing tool, CRISPR-Cas9, for treating neurodegenerative diseases.

    PubMed

    Kolli, Nivya; Lu, Ming; Maiti, Panchanan; Rossignol, Julien; Dunbar, Gary L

    2018-01-01

    Increased accumulation of transcribed protein from the damaged DNA and reduced DNA repair capability contributes to numerous neurological diseases for which effective treatments are lacking. Gene editing techniques provide new hope for replacing defective genes and DNA associated with neurological diseases. With advancements in using such editing tools as zinc finger nucleases (ZFNs), meganucleases, and transcription activator-like effector nucleases (TALENs), etc., scientists are able to design DNA-binding proteins, which can make precise double-strand breaks (DSBs) at the target DNA. Recent developments with the CRISPR-Cas9 gene-editing technology has proven to be more precise and efficient when compared to most other gene-editing techniques. Two methods, non-homologous end joining (NHEJ) and homology-direct repair (HDR), are used in CRISPR-Cas9 system to efficiently excise the defective genes and incorporate exogenous DNA at the target site. In this review article, we provide an overview of the CRISPR-Cas9 methodology, including its molecular mechanism, with a focus on how in this gene-editing tool can be used to counteract certain genetic defects associated with neurological diseases. Detailed understanding of this new tool could help researchers design specific gene editing strategies to repair genetic disorders in selective neurological diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Generation of megabase-scale deletions, inversions and duplications involving the Contactin-6 gene in mice by CRISPR/Cas9 technology.

    PubMed

    Korablev, Alexei N; Serova, Irina A; Serov, Oleg L

    2017-12-28

    Copy Number Variation (CNV) of the human CNTN6 gene (encoding the contactin-6 protein), caused by deletions or duplications, is responsible for severe neurodevelopmental impairments, often in combination with facial dysmorphias. Conversely, deleterious point mutations of this gene do not show any clinical phenotypes. The aim of this study is to generate mice carrying large deletions, duplications and inversions involving the Cntn6 gene as a new experimental model to study CNV of the human CNTN6 locus. To generate large chromosomal rearrangements on mouse chromosome 6, we applied CRISPR/Cas9 technology in zygotes. Two guide RNAs (gRNAs) (flanking a DNA fragment of 1137 Mb) together with Cas9 mRNA and single-stranded DNA oligonucleotides (ssODN) were microinjected into the cytoplasm of 599 zygotes of F1 (C57BL x CBA) mice, and 256 of them were transplanted into oviducts of CD-1 females. As a result, we observed the birth of 41 viable F0 offspring. Genotyping of these mice was performed by PCR analysis and sequencing of PCR products. Among the 41 F0 offspring, we identified seven mice with deletions, two animals carrying duplications of the gene and four carrying inversions. Interestingly, two F0 offspring had both deletions and duplications. It is important to note that while three of seven deletion carriers showed expected sequences at the new joint sites, in another three, we identified an absence of 1-10 nucleotides at the CRISPR/Cas9 cut sites, and in one animal, 103 bp were missing, presumably due to error-prone non-homologous end joining. In addition, we detected the absence of 5 and 13 nucleotides at these sites in two F0 duplication carriers. Similar sequence changes at CRISPR/Cas9 cut sites were observed at the right and left boundaries of inversions. Thus, megabase-scale deletions, duplications and inversions were identified in 11 F0 offspring among 41 analyzed, i.e., approximately 25% efficiency. All genetically modified F0 offspring were viable and

  18. Corrective Action Investigation Plan for Corrective Action Unit 140: Waste Dumps, Burn Pits, and Storage Area, Nevada Test Site, Nevada, July 2002, Rev. No. 0

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NNSA /NV

    This Corrective Action Investigation Plan contains the U.S. Department of Energy, National Nuclear Security Administration Nevada Operations Office's approach to collect the data necessary to evaluate corrective action alternatives appropriate for the closure of Corrective Action Unit (CAU) 140 under the Federal Facility Agreement and Consent Order. Corrective Action Unit 140 consists of nine Corrective Action Sites (CASs): 05-08-01, Detonation Pits; 05-08-02, Debris Pits; 05-17-01, Hazardous Waste Accumulation Site (Buried); 05-19-01, Waste Disposal Site; 05-23-01, Gravel Gertie; 05-35-01, Burn Pit; 05-99-04, Burn Pit; 22-99-04, Radioactive Waste Dump; 23-17-01, Hazardous Waste Storage Area. All nine of these CASs are located withinmore » Areas 5, 22, and 23 of the Nevada Test Site (NTS) in Nevada, approximately 65 miles northwest of Las Vegas. This CAU is being investigated because disposed waste may be present without appropriate controls (i.e., use restrictions, adequate cover) and hazardous and/or radioactive constituents may be present or migrating at concentrations and locations that could potentially pose a threat to human health and the environment. The NTS has been used for various research and development projects including nuclear weapons testing. The CASs in CAU 140 were used for testing, material storage, waste storage, and waste disposal. A two-phase approach has been selected to collect information and generate data to satisfy needed resolution criteria and resolve the decision statements. Phase I will determine if contaminants of potential concern (COPCs) are present in concentrations exceeding preliminary action levels. This data will be evaluated at all CASs. Phase II will determine the extent of the contaminant(s) of concern (COCs). This data will only be evaluated for CASs with a COC identified during Phase I. Based on process knowledge, the COPCs for CAU 140 include volatile organics, semivolatile organics, petroleum hydrocarbons, explosive

  19. Efficient CRISPR/Cas9-based gene knockout in watermelon.

    PubMed

    Tian, Shouwei; Jiang, Linjian; Gao, Qiang; Zhang, Jie; Zong, Mei; Zhang, Haiying; Ren, Yi; Guo, Shaogui; Gong, Guoyi; Liu, Fan; Xu, Yong

    2017-03-01

    CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

  20. [CAS in rhino-surgical procedures in the growing age].

    PubMed

    Schipper, J; Maier, W; Gellrich, N-C; Arapakis, I; Hochmuth, A; Laszig, R

    2005-01-01

    Rhinosurgery in children and adolescents meets special requirements: Limited cooperation and reduced limits for the organ dose for ionizing radiological examinations aggravate diagnostics. On the other side, bone sutures and bone growth areas have to be respected intraoperatively, and regions of bones not yet calcified have to be distinguished from possible tumor infiltration. Computer assisted surgery (CAS) can help to identify these areas safely. 5 patients, from the first to the 20 (th) year of life, suffering from tumors, malformation syndromes or therapy resistant nasal polyposis were treated with CAS in rhinosurgery. In addition to radiological diagnostics, we performed 3D computed tomography of the skull for CAS. CAS enabled us to intraoperatively respect possible areas of bone growth, to identify regions with thin, not bonily developed cranial vault and to safely distinguish bone sutures from ethmoidal cells. CAS helped the surgeon to navigate in the not yet developed paranasal sinus system. CAS is a useful complementary method in rhinosurgery of the developing skull of the child. In spite of the additional 3D computed tomography, the calculated organ dose of the ocular lense amounted to 5 millisievert, so a recommended maximal organ dose for the ocular lense of 15 millisievert was not exceeded.