21 CFR 184.1033 - Citric acid.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Citric acid. 184.1033 Section 184.1033 Food and... Substances Affirmed as GRAS § 184.1033 Citric acid. (a) Citric acid (C6H8O7, CAS Reg. No. 77-92-9) is the... mole of water per mole of citric acid. Citric acid may be produced by recovery from sources such as...

40 CFR 180.910 - Inert ingredients used pre- and post-harvest; exemptions from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

..., carrier Lactic acid Solvent Lactic acid, 2-ethylhexyl ester (CAS Reg. No. 6283-86-9) Solvent Lactic acid, 2-ethylhexyl ester, (2S)- (CAS Reg. No. 186817-80-1) Solvent Lactic acid, n-propyl ester, (S); (CAS... coating agent Petroleum wax, conforming to 21 CFR 172.886(d) Coating agent Phosphoric acid Buffer...

21 CFR 184.1033 - Citric acid.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Citric acid. 184.1033 Section 184.1033 Food and....1033 Citric acid. (a) Citric acid (C6H8O7, CAS Reg. No. 77-92-9) is the compound 2-hydroxy-1,2,3... crystals or a white powder and may be anhydrous or contain one mole of water per mole of citric acid...

21 CFR 184.1061 - Lactic acid.

Code of Federal Regulations, 2014 CFR

2014-04-01

....1061 Lactic acid. (a) Lactic acid (C3H6O3, CAS Reg. Nos.: dl mixture, 598-82-3; l-isomer, 79-33-4; d... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Lactic acid. 184.1061 Section 184.1061 Food and... hydrolysis to lactic acid. (b) The ingredient meets the specifications of the Food Chemicals Codex, 3d Ed...

21 CFR 184.1099 - Tartaric acid.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Tartaric acid. 184.1099 Section 184.1099 Food and... Substances Affirmed as GRAS § 184.1099 Tartaric acid. (a) Food grade tartaric acid (C4H6O6, CAS Reg. No. 87-69-4) has the l configuration. The l form of tartaric acid is dextrorotatory in solution and is also...

21 CFR 175.320 - Resinous and polymeric coatings for polyolefin films.

Code of Federal Regulations, 2010 CFR

2010-04-01

...-formaldehyde as the basic polymer or chemically modified with methyl alcohol Methacrylic acid and its ethyl or... 68083-18-1), with methyl hydrogen polysiloxane (CAS Reg. No. 63148-57-2). Dimethyl maleate (CAS Reg. No... product of vinyl-containing dimethylpolysiloxane (CAS Reg. Nos. 68083-19-2 and 68083-18-1), with methyl...

21 CFR 175.320 - Resinous and polymeric coatings for polyolefin films.

Code of Federal Regulations, 2011 CFR

2011-04-01

...-formaldehyde as the basic polymer or chemically modified with methyl alcohol Methacrylic acid and its ethyl or... 68083-18-1), with methyl hydrogen polysiloxane (CAS Reg. No. 63148-57-2). Dimethyl maleate (CAS Reg. No... product of vinyl-containing dimethylpolysiloxane (CAS Reg. Nos. 68083-19-2 and 68083-18-1), with methyl...

21 CFR 184.1069 - Malic acid.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., CAS Reg. No. of DL-form 617-48-1) is the common name for 1-hydroxy-1, 2-ethanedicarboxylic acid. L... ingredients are used in food, except baby food, at levels not to exceed good manufacturing practice in accordance with § 184.1(b)(1). Current good manufacturing practice results in a maximum level, as served, of...

21 CFR 184.1069 - Malic acid.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., CAS Reg. No. of DL-form 617-48-1) is the common name for 1-hydroxy-1, 2-ethanedicarboxylic acid. L... ingredients are used in food, except baby food, at levels not to exceed good manufacturing practice in accordance with § 184.1(b)(1). Current good manufacturing practice results in a maximum level, as served, of...

21 CFR 178.3725 - Pigment dispersants.

Code of Federal Regulations, 2010 CFR

2010-04-01

.... Phosphorylated tall oil fatty acids (CAS Reg. No. 68604-99-9), prepared by the reaction of dimethyl hydrogen phosphite with tall oil fatty acids For use only at levels not to exceed 1.0 percent by weight of the... materials. Substances Limitations Dimethylolpropionic acid (CAS Reg. No. 4767-03-7) For use only at levels...

75 FR 70236 - Hop Beta Acids; Receipt of Application for Emergency Exemption, Solicitation of Public Comment

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-17

... of Agriculture to use hop beta acids (CAS Reg. No. none specified) to treat up to 181,000 honey bee... exemption regional request for use of hop beta acids in honey bee hives to control varroa mites. Information... effect on honey bee populations. The parasitic mite is considered the primary pest of honeybees and its...

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.

Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

PubMed Central

Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Kellner, Max J.; Joung, Julia; Collins, James J.; Zhang, Feng

2018-01-01

Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) that combines isothermal pre-amplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCKv2: 1) 4-channel single reaction multiplexing using orthogonal CRISPR enzymes; 2) quantitative measurement of input down to 2 aM; 3) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxilary CRISPR-associated enzyme; and 4) lateral flow read-out. SHERLOCKv2 can detect Dengue or Zika virus ssRNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids. PMID:29449508

Mechanical behavior of glass and Blackglas{reg_sign} ceramic matrix composite

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

Stawovy, R.H.; Kampe, S.L.; Curtin, W.A.

Room temperature tensile tests are reported on two low-cost ceramic matrix composite materials, comprised of matrices of Blackglas{reg_sign} and a proprietary glass composition each reinforced with Nicalon{reg_sign} SiC-based fibers. The measured mechanical behaviors, supplemented by post-fracture analysis of fiber pullout and fiber fracture mirrors, are compared in detail to the performance predicted theoretically. This allows for an assessment of the roles of the matrix, fiber strength, residual stresses, fiber geometry, and the fiber/matrix interfacial properties in determining mechanical response. The Blackglas{reg_sign} matrix cracks extensively during processing, and so the mechanical response is controlled by the deformation and fracture of themore » fiber bundle. The interfacial sliding resistance, {tau}, is determined to be {approx} 17 MPa and the in-situ (post-processed) fiber characteristic strength, {sigma}{sub c} is found to be {approx} 2.0 GPa, both similar to values reported in the literature for Nicalon{reg_sign}/CAS-glass systems. For the glass matrix, the unidirectional and cross-ply materials show marked differences in mechanical behavior. In the cross-ply composites, {tau} {approx} 14 MPa and {sigma}{sub c} {approx} 2.9 GPa; in the unidirectional variants, these values were 1.7 MPa and 1.6 GPa, respectively. With these data and other derived micromechanical parameters, the stress-strain and failure point of these materials was predicted using existing models, and excellent agreement with the experiments was obtained. These materials thus perform as expected given the in-situ fiber and interface properties. Notably, the cross-ply glass matrix composites exhibit high fiber strength retention and hence show tensile strengths that are better than other Nicalon{reg_sign}-based materials tested to date.« less

Pilot test of Pickliq{reg_sign} process to determine energy and environmental benefits & economic feasibility

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

Olsen, D.R.

Green Technology Group (GTG) was awarded Grant No. DE-FG01-96EE 15657 in the amount of $99,904 for a project to advance GTG`s Pickliq{reg_sign} Process in the Copper and Steel Industries. The use of the Pickliq{reg_sign} Process can significantly reduce the production of waste acids containing metal salts. The Pickliq{reg_sign} Process can save energy and eliminate hazardous waste in a typical copper rod or wire mill or a typical steel wire mill. The objective of this pilot project was to determine the magnitude of the economic, energy and environmental benefits of the Pickliq{reg_sign} Process in two applications within the metal processing industry.more » The effectiveness of the process has already been demonstrated at facilities cleaning iron and steel with sulfuric acid. 9207 companies are reported to use sulfuric and hydrochloric acid in the USA. The USEPA TRI statistics of acid not recycled in the US is 2.4 x 10{sup 9} lbs (net) for Hydrochloric Acid and 2.0 x 10{sup 9} lbs (net) for Sulfuric Acid. The energy cost of not reclaiming acid is 10.7 x 10{sup 6} BTU/ton for Hydrochloric Acid and 21.6 x 10{sup 6} BTU/Ton for Sulfuric Acid. This means that there is a very large market for the application of the Pickliq{reg_sign} Process and the widespread use of the process will bring significant world wide savings of energy to the environment.« less

The expression of REG 1A and REG 1B is increased during acute amebic colitis.

PubMed

Peterson, Kristine M; Guo, Xiaoti; Elkahloun, Abdel G; Mondal, Dinesh; Bardhan, Pradip K; Sugawara, Akira; Duggal, Priya; Haque, Rashidul; Petri, William A

2011-09-01

Entamoeba histolytica, a protozoan parasite, is an important cause of diarrhea and colitis in the developing world. Amebic colitis is characterized by ulceration of the intestinal mucosa. We performed microarray analysis of intestinal biopsies during acute and convalescent amebiasis in order to identify genes potentially involved in tissue injury or repair. Colonic biopsy samples were obtained from 8 patients during acute E. histolytica colitis and again 60 days after recovery. Gene expression in the biopsies was evaluated using microarray, and confirmed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). REG 1A and REG 1B were the most up-regulated of all genes in the human intestine in acute versus convalescent E. histolytica disease: as determined by microarray, the levels of induction were 7.4-fold and 10.7 fold for REG 1A and B; p=0.003 and p=0.006 respectively. Increased expression of REG 1A and REG 1B protein in the colonic crypt epithelial cells during acute amebiasis was similarly observed by immunohistochemistry. Because REG 1 protein is anti-apoptotic and pro-proliferative, and since E. histolytica induces apoptosis of the intestinal epithelium as part of its disease process, we next tested if REG 1 might be protective during amebiasis by preventing parasite-induced apoptosis. Intestinal epithelial cells from REG 1-/- mice were found to be more susceptible to spontaneous, and parasite-induced, apoptosis in vitro (p=0.03). We concluded that REG 1A and REG 1B were upregulated during amebiasis and may function to protect the intestinal epithelium from parasite-induced apoptosis. Published by Elsevier Ireland Ltd.

21 CFR 184.1721 - Sodium acetate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium acetate. 184.1721 Section 184.1721 Food and....1721 Sodium acetate. (a) Sodium acetate (C2H3O2Na, CAS Reg. No. 127-09-3 or C2H3O2Na·3H2O, CAS Reg. No. 6131-90-4) is the sodium salt of acetic acid and occurs naturally in plant and animal tissues. Sodium...

The Global Redox Responding RegB/RegA Signal Transduction System Regulates the Genes Involved in Ferrous Iron and Inorganic Sulfur Compound Oxidation of the Acidophilic Acidithiobacillus ferrooxidans.

PubMed

Moinier, Danielle; Byrne, Deborah; Amouric, Agnès; Bonnefoy, Violaine

2017-01-01

The chemical attack of ore by ferric iron and/or sulfuric acid releases valuable metals. The products of these reactions are recycled by iron and sulfur oxidizing microorganisms. These acidophilic chemolithotrophic prokaryotes, among which Acidithiobacillus ferrooxidans , grow at the expense of the energy released from the oxidation of ferrous iron and/or inorganic sulfur compounds (ISCs). In At. ferrooxidans , it has been shown that the expression of the genes encoding the proteins involved in these respiratory pathways is dependent on the electron donor and that the genes involved in iron oxidation are expressed before those responsible for ISCs oxidation when both iron and sulfur are present. Since the redox potential increases during iron oxidation but remains stable during sulfur oxidation, we have put forward the hypothesis that the global redox responding two components system RegB/RegA is involved in this regulation. To understand the mechanism of this system and its role in the regulation of the aerobic respiratory pathways in At. ferrooxidans , the binding of different forms of RegA (DNA binding domain, wild-type, unphosphorylated and phosphorylated-like forms of RegA) on the regulatory region of different genes/operons involved in ferrous iron and ISC oxidation has been analyzed. We have shown that the four RegA forms are able to bind specifically the upstream region of these genes. Interestingly, the phosphorylation of RegA did not change its affinity for its cognate DNA. The transcriptional start site of these genes/operons has been determined. In most cases, the RegA binding site(s) was (were) located upstream from the -35 (or -24) box suggesting that RegA does not interfere with the RNA polymerase binding. Based on the results presented in this report, the role of the RegB/RegA system in the regulation of the ferrous iron and ISC oxidation pathways in At. ferrooxidans is discussed.

The Global Redox Responding RegB/RegA Signal Transduction System Regulates the Genes Involved in Ferrous Iron and Inorganic Sulfur Compound Oxidation of the Acidophilic Acidithiobacillus ferrooxidans

PubMed Central

Moinier, Danielle; Byrne, Deborah; Amouric, Agnès; Bonnefoy, Violaine

2017-01-01

The chemical attack of ore by ferric iron and/or sulfuric acid releases valuable metals. The products of these reactions are recycled by iron and sulfur oxidizing microorganisms. These acidophilic chemolithotrophic prokaryotes, among which Acidithiobacillus ferrooxidans, grow at the expense of the energy released from the oxidation of ferrous iron and/or inorganic sulfur compounds (ISCs). In At. ferrooxidans, it has been shown that the expression of the genes encoding the proteins involved in these respiratory pathways is dependent on the electron donor and that the genes involved in iron oxidation are expressed before those responsible for ISCs oxidation when both iron and sulfur are present. Since the redox potential increases during iron oxidation but remains stable during sulfur oxidation, we have put forward the hypothesis that the global redox responding two components system RegB/RegA is involved in this regulation. To understand the mechanism of this system and its role in the regulation of the aerobic respiratory pathways in At. ferrooxidans, the binding of different forms of RegA (DNA binding domain, wild-type, unphosphorylated and phosphorylated-like forms of RegA) on the regulatory region of different genes/operons involved in ferrous iron and ISC oxidation has been analyzed. We have shown that the four RegA forms are able to bind specifically the upstream region of these genes. Interestingly, the phosphorylation of RegA did not change its affinity for its cognate DNA. The transcriptional start site of these genes/operons has been determined. In most cases, the RegA binding site(s) was (were) located upstream from the −35 (or −24) box suggesting that RegA does not interfere with the RNA polymerase binding. Based on the results presented in this report, the role of the RegB/RegA system in the regulation of the ferrous iron and ISC oxidation pathways in At. ferrooxidans is discussed. PMID:28747899

21 CFR 184.1721 - Sodium acetate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium acetate. 184.1721 Section 184.1721 Food and... Substances Affirmed as GRAS § 184.1721 Sodium acetate. (a) Sodium acetate (C2H3O2Na, CAS Reg. No. 127-09-3 or C2H3O2Na·3H2O, CAS Reg. No. 6131-90-4) is the sodium salt of acetic acid and occurs naturally in plant and...

21 CFR 184.1721 - Sodium acetate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium acetate. 184.1721 Section 184.1721 Food and... Substances Affirmed as GRAS § 184.1721 Sodium acetate. (a) Sodium acetate (C2H3O2Na, CAS Reg. No. 127-09-3 or C2H3O2Na·3H2O, CAS Reg. No. 6131-90-4) is the sodium salt of acetic acid and occurs naturally in plant and...

21 CFR 184.1721 - Sodium acetate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium acetate. 184.1721 Section 184.1721 Food and... Substances Affirmed as GRAS § 184.1721 Sodium acetate. (a) Sodium acetate (C2H3O2Na, CAS Reg. No. 127-09-3 or C2H3O2Na·3H2O, CAS Reg. No. 6131-90-4) is the sodium salt of acetic acid and occurs naturally in plant and...

21 CFR 184.1721 - Sodium acetate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium acetate. 184.1721 Section 184.1721 Food and... Substances Affirmed as GRAS § 184.1721 Sodium acetate. (a) Sodium acetate (C2H3O2Na, CAS Reg. No. 127-09-3 or C2H3O2Na·3H2O, CAS Reg. No. 6131-90-4) is the sodium salt of acetic acid and occurs naturally in plant and...

T-Reg Comparator: an analysis tool for the comparison of position weight matrices

PubMed Central

Roepcke, Stefan; Grossmann, Steffen; Rahmann, Sven; Vingron, Martin

2005-01-01

T-Reg Comparator is a novel software tool designed to support research into transcriptional regulation. Sequence motifs representing transcription factor binding sites are usually encoded as position weight matrices. The user inputs a set of such weight matrices or binding site sequences and our program matches them against the T-Reg database, which is presently built on data from the Transfac [E. Wingender (2004) In Silico Biol., 4, 55–61] and Jaspar [A. Sandelin, W. Alkema, P. Engstrom, W. W. Wasserman and B. Lenhard (2004) Nucleic Acids Res., 32, D91–D94]. Our tool delivers a detailed report on similarities between user-supplied motifs and motifs in the database. Apart from simple one-to-one relationships, T-Reg Comparator is also able to detect similarities between submatrices. In addition, we provide a user interface to a program for sequence scanning with weight matrices. Typical areas of application for T-Reg Comparator are motif and regulatory module finding and annotation of regulatory genomic regions. T-Reg Comparator is available at . PMID:15980506

T-Reg Comparator: an analysis tool for the comparison of position weight matrices.

PubMed

Roepcke, Stefan; Grossmann, Steffen; Rahmann, Sven; Vingron, Martin

2005-07-01

T-Reg Comparator is a novel software tool designed to support research into transcriptional regulation. Sequence motifs representing transcription factor binding sites are usually encoded as position weight matrices. The user inputs a set of such weight matrices or binding site sequences and our program matches them against the T-Reg database, which is presently built on data from the Transfac [E. Wingender (2004) In Silico Biol., 4, 55-61] and Jaspar [A. Sandelin, W. Alkema, P. Engstrom, W. W. Wasserman and B. Lenhard (2004) Nucleic Acids Res., 32, D91-D94]. Our tool delivers a detailed report on similarities between user-supplied motifs and motifs in the database. Apart from simple one-to-one relationships, T-Reg Comparator is also able to detect similarities between submatrices. In addition, we provide a user interface to a program for sequence scanning with weight matrices. Typical areas of application for T-Reg Comparator are motif and regulatory module finding and annotation of regulatory genomic regions. T-Reg Comparator is available at http://treg.molgen.mpg.de.

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.

21 CFR 73.3121 - Poly(hydroxyethyl methacrylate)-dye copolymers.

Code of Federal Regulations, 2011 CFR

2011-04-01

...-naphthalenedisulfonic acid, 4-amino-5-hydroxy-3,6-bis((4-((2-(sulfooxy)ethyl)sulfonyl)phenyl)azo)-tetrasodium salt] (CAS...)-] (CAS Reg. No. 60958-41-0); (5) Reactive Blue No. 19 [2-anthracene-sulfonic acid, 1-amino-9,10-dihydro-9...); (6) Reactive Blue No. 4 [2-anthracenesulfonic acid, 1-amino-4-(3-((4,6-dichloro-s-triazin-2-yl)amino...

21 CFR 73.3121 - Poly(hydroxyethyl methacrylate)-dye copolymers.

Code of Federal Regulations, 2010 CFR

2010-04-01

...-naphthalenedisulfonic acid, 4-amino-5-hydroxy-3,6-bis((4-((2-(sulfooxy)ethyl)sulfonyl)phenyl)azo)-tetrasodium salt] (CAS...)-] (CAS Reg. No. 60958-41-0); (5) Reactive Blue No. 19 [2-anthracene-sulfonic acid, 1-amino-9,10-dihydro-9...); (6) Reactive Blue No. 4 [2-anthracenesulfonic acid, 1-amino-4-(3-((4,6-dichloro-s-triazin-2-yl)amino...

21 CFR 177.1632 - Poly (phenyl-enetereph-thala-mide) resins.

Code of Federal Regulations, 2010 CFR

2010-04-01

...). Poly(oxyethylene) sorbitol monolaurate tetraoleate (CAS Reg. No. 71243-28-2). Poly(oxyethylene) sorbitol hexaoleate (CAS Reg. No. 57171-56-9). 4,4′-Butylidenebis (6-tert-butyl-m-cresol) (CAS Reg. No. 85...

21 CFR 184.1195 - Calcium citrate.

Code of Federal Regulations, 2012 CFR

2012-04-01

..., CAS Reg. No. 813-0994-095) is the calcium salt of citric acid. It is prepared by neutralizing citric acid with calcium hydroxide or calcium carbonate. It occurs as a fine white, odorless powder and...

21 CFR 184.1195 - Calcium citrate.

Code of Federal Regulations, 2013 CFR

2013-04-01

..., CAS Reg. No. 813-0994-095) is the calcium salt of citric acid. It is prepared by neutralizing citric acid with calcium hydroxide or calcium carbonate. It occurs as a fine white, odorless powder and...

Development of Thixomolded{reg_sign} magnesium products

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

Ghosh, D.; Fan, R.; Kang, K.

1995-10-01

Thixomolding{reg_sign} is a racial new process which merges the technologies of die-casting and plastic injection molding for the net shape molding of magnesium based alloys. Properties of Thixomolded{reg_sign} magnesium alloys are discussed and compared with those of traditional die casting. Magnesium alloys are of great interest to automobile manufacturers because of the potential weight savings and corresponding energy savings due to increased fuel economy. For this reason, one of the first target markets for Thixomolded{reg_sign} products is the automotive industry. The use of Thixomolding{reg_sign} in the production of an automobile part is examined.

21 CFR 184.1440 - Magnesium stearate.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., CAS Reg. No. 557-04-0) is the magnesium salt of stearic acid. It is produced as a white precipitate by... derived from stearic acid that is obtained from edible sources and that conforms to the requirements of...

21 CFR 184.1440 - Magnesium stearate.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., CAS Reg. No. 557-04-0) is the magnesium salt of stearic acid. It is produced as a white precipitate by... derived from stearic acid that is obtained from edible sources and that conforms to the requirements of...

21 CFR 178.3725 - Pigment dispersants.

Code of Federal Regulations, 2011 CFR

2011-04-01

.... Phosphorylated tall oil fatty acids (CAS Reg. No. 68604-99-9), prepared by the reaction of dimethyl hydrogen phosphite with tall oil fatty acids For use only at levels not to exceed 1.0 percent by weight of the...

40 CFR 180.920 - Inert ingredients used pre-harvest; exemptions from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Defoamer γ-Butyrolactone Solvent C.I. Pigment Blue #15 (CAS Reg. No. 147-14-8; containing no more than 50... Surfactants, related adjuvants of surfactants Aluminum sulfate Safener adjuvant Ammonium chloride (CAS Reg. No... herbicides Ammonium polyphosphate (CAS Reg. No. 68333-79-9) Sequestrant, buffer, or surfactant Barium sulfate...

40 CFR 180.930 - Inert ingredients applied to animals; exemptions from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... clay Solid diluent, carrier Barium sulfate (CAS Reg. No. 7727-43-7) Carrier, density control agent...-Difluoroethane (CAS Reg. No. 75-37-6) For aerosol pesticide formulations used for insect control in food- and... tris-12-hydroxystearate Flow control agent Graphite Solid diluent, carrier n-Hexyl alcohol (CAS Reg. No...

BP Reg Experiment Operations

NASA Image and Video Library

2015-04-07

ISS043E091755 (04/07/2015) --- Expedition 43 Commander Terry Virts is seen here working inside of the Columbus laboratory on the Blood Pressure Regulation (BP Reg) experiment. Astronauts returning from long-duration space flights risk experiencing dizziness or fainting when they stand immediately after returning to Earth. This has an important health risk as it reduces the potential for astronauts to safely escape from an emergency situation. BP Reg will help researchers develop appropriate countermeasures so that astronauts returning from long-duration space flights will have very low risk of experiencing dizziness or fainting when they return to Earth.

BP Reg Experiment Operations

NASA Image and Video Library

2015-04-07

ISS043E091740 (04/07/2015) --- Expedition 43 Commander Terry Virts is seen here working inside of the Columbus laboratory on the Blood Pressure Regulation (BP Reg) experiment. Astronauts returning from long-duration space flights risk experiencing dizziness or fainting when they stand immediately after returning to Earth. This has an important health risk as it reduces the potential for astronauts to safely escape from an emergency situation. BP Reg will help researchers develop appropriate countermeasures so that astronauts returning from long-duration space flights will have very low risk of experiencing dizziness or fainting when they return to Earth.

21 CFR 178.1010 - Sanitizing solutions.

Code of Federal Regulations, 2010 CFR

2010-04-01

... permanganate (CAS Reg. No. 7722-64-7). Magnesium oxide (CAS Reg. No. 1309-48-4) and potassium bromide (CAS Reg... permanganate. (ii) The solution identified in paragraph (b)(37) of this section with potassium bromide shall... potassium permanganate. (iii) Magnesium oxide when used in paragraph (c)(32) (i) or (ii) of this section...

[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.

Epigenetic regulation of REG1A and chemosensitivity of cutaneous melanoma

PubMed Central

Sato, Yusuke; Marzese, Diego M; Ohta, Katsuya; Huang, Sharon K; Sim, Myung Shin; Chong, Kelly; Hoon, Dave SB

2013-01-01

Regenerating gene 1A (REG1A) plays an important role in tissue regeneration and in cell proliferation in epithelium origin tumors; however, its role in melanoma has not been explored in details. The objective of this study was to identify whether REG1A is expressed in cutaneous melanoma and if REG1A expression status can predict prognosis in cutaneous melanoma patients with metastasis. We also determined whether epigenetic regulation of the promoter region regulates REG1A expression. AJCC stage III cutaneous melanoma specimens with clinically well annotated stage III lymph node melanoma metastasis tissue microarray were assessed by IHC. MALDI-TOF-mass spectrometry and HM450K array were used to identify REG1A promoter region CpG site methylation. Chemotherapeutic agent response by melanoma cells as related to REG1A protein expression was assessed. Post-surgery melanoma patients followed by adjuvant chemotherapy with high REG1A expression had a significantly better prognosis (disease-specific survival) compared with patients with low REG1A expression (log rank test; p = 0.0013). The demethylating reagent 5-Aza-2′-deoxycytidine activated REG1A promoter region resulting in enhanced REG1A mRNA and protein expression in melanoma cell lines. Promoter region CpG methylation was shown to regulate REG1A expression in melanoma cells. Moreover, melanoma lines with high REG1A mRNA expression were more susceptible to Dacarbazine and Cisplatin, as compared with those with low REG1A mRNA expression. In conclusion, REG1A expression status may be useful as a biomarker in melanoma patients for sensitivity to these chemotherapeutic agents. The epigenetic regulation of the REG1A promoter region may offer a potential therapeutic approach to improve chemotherapy for metastatic melanoma patients. PMID:23903855

CatReg Software for Categorical Regression Analysis (May 2016)

EPA Science Inventory

CatReg 3.0 is a Microsoft Windows enhanced version of the Agency’s categorical regression analysis (CatReg) program. CatReg complements EPA’s existing Benchmark Dose Software (BMDS) by greatly enhancing a risk assessor’s ability to determine whether data from separate toxicologic...

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

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.

21 CFR 184.1140 - Ammonium citrate, dibasic.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., dibasic ((NH4)2HC6H5O7, CAS Reg. No. 3012-65-5) is the diammonium salt of citric acid. It is prepared by partially neutralizing citric acid with ammonia. (b) The ingredient must be of a purity suitable for its...

21 CFR 184.1140 - Ammonium citrate, dibasic.

Code of Federal Regulations, 2012 CFR

2012-04-01

..., dibasic ((NH4)2HC6H5O7, CAS Reg. No. 3012-65-5) is the diammonium salt of citric acid. It is prepared by partially neutralizing citric acid with ammonia. (b) The ingredient must be of a purity suitable for its...

21 CFR 184.1140 - Ammonium citrate, dibasic.

Code of Federal Regulations, 2013 CFR

2013-04-01

..., dibasic ((NH4)2HC6H5O7, CAS Reg. No. 3012-65-5) is the diammonium salt of citric acid. It is prepared by partially neutralizing citric acid with ammonia. (b) The ingredient must be of a purity suitable for its...

21 CFR 184.1140 - Ammonium citrate, dibasic.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., dibasic ((NH4)2HC6H5O7, CAS Reg. No. 3012-65-5) is the diammonium salt of citric acid. It is prepared by partially neutralizing citric acid with ammonia. (b) The ingredient must be of a purity suitable for its...

21 CFR 184.1207 - Calcium lactate.

Code of Federal Regulations, 2014 CFR

2014-04-01

..., CAS Reg. No. 814-80-2) is prepared commercially by the neutralization of lactic acid with calcium.... (d) Prior sanctions for this ingredient different from the uses established in this section do not...

21 CFR 74.1306 - D&C Red No. 6.

Code of Federal Regulations, 2011 CFR

2011-04-01

...-naphthalenecarboxylic acid (CAS Reg. No. 5858-81-1). To manufacture the additive, 2-amino-5-methylbenzenesulfonic acid... an appendix A to part 74. 2-Amino-5-methylbenzenesulfonic acid, sodium salt, not more than 0.2... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR...

21 CFR 74.705 - FD&C Yellow No. 5.

Code of Federal Regulations, 2010 CFR

2010-04-01

...-sulfophenyl-azo]-1H-pyrazole-3-carboxylic acid (CAS Reg. No. 1934-21-0). To manufacture the additive, 4-amino-benzenesulfonic acid is diazotized using hydrochloric acid and sodium nitrite. The diazo compound is coupled with... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR...

21 CFR 74.1306 - D&C Red No. 6.

Code of Federal Regulations, 2010 CFR

2010-04-01

...-naphthalenecarboxylic acid (CAS Reg. No. 5858-81-1). To manufacture the additive, 2-amino-5-methylbenzenesulfonic acid... an appendix A to part 74. 2-Amino-5-methylbenzenesulfonic acid, sodium salt, not more than 0.2... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR...

21 CFR 74.705 - FD&C Yellow No. 5.

Code of Federal Regulations, 2011 CFR

2011-04-01

...-sulfophenyl-azo]-1H-pyrazole-3-carboxylic acid (CAS Reg. No. 1934-21-0). To manufacture the additive, 4-amino-benzenesulfonic acid is diazotized using hydrochloric acid and sodium nitrite. The diazo compound is coupled with... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR...

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.

21 CFR 74.1307 - D&C Red No. 7.

Code of Federal Regulations, 2010 CFR

2010-04-01

...-naphthalenecarboxylic acid (CAS Reg. No. 5281-04-9). To manufacture the additive, 2-amino-5-methylbenzenesulfonic acid.... 6 and 7,” which is an appendix A to part 74. 2-Amino-5-methylbenzenesulfonic acid, calcium salt, not... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR...

21 CFR 74.1307 - D&C Red No. 7.

Code of Federal Regulations, 2011 CFR

2011-04-01

...-naphthalenecarboxylic acid (CAS Reg. No. 5281-04-9). To manufacture the additive, 2-amino-5-methylbenzenesulfonic acid.... 6 and 7,” which is an appendix A to part 74. 2-Amino-5-methylbenzenesulfonic acid, calcium salt, not... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR...

21 CFR 184.1323 - Glyceryl monooleate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... prepared by esterification of commerical oleic acid that is derived either from edible sources or from tall oil fatty acids meeting the requirements of § 172.862 of this chapter. It contains glyceryl monooleate (C21H40O4, CAS Reg. No. 25496-72-4) and glyceryl esters of fatty acids present in commercial oleic acid. (b...

21 CFR 184.1323 - Glyceryl monooleate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... prepared by esterification of commerical oleic acid that is derived either from edible sources or from tall oil fatty acids meeting the requirements of § 172.862 of this chapter. It contains glyceryl monooleate (C21H40O4, CAS Reg. No. 25496-72-4) and glyceryl esters of fatty acids present in commercial oleic acid. (b...

21 CFR 184.1323 - Glyceryl monooleate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... prepared by esterification of commerical oleic acid that is derived either from edible sources or from tall oil fatty acids meeting the requirements of § 172.862 of this chapter. It contains glyceryl monooleate (C21H40O4, CAS Reg. No. 25496-72-4) and glyceryl esters of fatty acids present in commercial oleic acid. (b...

21 CFR 184.1207 - Calcium lactate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... integer up to 5, CAS Reg. No. 814-80-2) is prepared commercially by the neutralization of lactic acid with.... (d) Prior sanctions for this ingredient different from the uses established in this section do not...

21 CFR 74.1205 - D&C Green No. 5.

Code of Federal Regulations, 2010 CFR

2010-04-01

... percent. 1,4-Dihydroxyanthraquinone, not more than 0.2 percent. 2-Amino-m-toluenesulfonic acid, not more... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR...)diimino]bis-[5-methylbenzenesulfonic acid] (CAS Reg. No. 4403-90-1). (2) Color additive mixtures for use...

21 CFR 74.1205 - D&C Green No. 5.

Code of Federal Regulations, 2011 CFR

2011-04-01

... percent. 1,4-Dihydroxyanthraquinone, not more than 0.2 percent. 2-Amino-m-toluenesulfonic acid, not more... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR...)diimino]bis-[5-methylbenzenesulfonic acid] (CAS Reg. No. 4403-90-1). (2) Color additive mixtures for use...

21 CFR 175.360 - Vinylidene chloride copolymer coatings for nylon film.

Code of Federal Regulations, 2014 CFR

2014-04-01

... is applied as a continuous film over one or both sides of a base film produced from nylon resins... produced by copolymerizing vinylidene chloride with one or more of the monomers acrylic acid, acrylonitrile, ethyl acrylate, methacrylic acid, methyl acrylate, methyl methacrylate (CAS Reg. No. 80-62-6; maximum...

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.

Thiazolidinediones inhibit REG I{alpha} gene transcription in gastrointestinal cancer cells

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

Yamauchi, Akiyo; Laboratory of Molecular Genetics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578; Department of Biochemistry, Nara Medical University, Kashihara 634-8521

2009-02-13

REG (Regenerating gene) I{alpha} protein functions as a growth factor for gastrointestinal cancer cells, and its mRNA expression is strongly associated with a poor prognosis in gastrointestinal cancer patients. We here demonstrated that PPAR{gamma}-agonist thiazolidinediones (TZDs) inhibited cell proliferation and REG I{alpha} protein/mRNA expression in gastrointestinal cancer cells. TZDs inhibited the REG I{alpha} gene promoter activity, via its cis-acting element which lacked PPAR response element and could not bind to PPAR{gamma}, in PPAR{gamma}-expressing gastrointestinal cancer cells. The inhibition was reversed by co-treatment with a specific PPAR{gamma}-antagonist GW9662. Although TZDs did not inhibit the REG I{alpha} gene promoter activity in PPAR{gamma}-non-expressingmore » cells, PPAR{gamma} overexpression in the cells recovered their inhibitory effect. Taken together, TZDs inhibit REG I{alpha} gene transcription through a PPAR{gamma}-dependent pathway. The TZD-induced REG I{alpha} mRNA reduction was abolished by cycloheximide, indicating the necessity of novel protein(s) synthesis. TZDs may therefore be a candidate for novel anti-cancer drugs for patients with gastrointestinal cancer expressing both REG I{alpha} and PPAR{gamma}.« less

21 CFR 172.846 - Sodium stearoyl lactylate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium stearoyl lactylate. 172.846 Section 172.846... Sodium stearoyl lactylate. The food additive sodium stearoyl lactylate (CAS Reg. No. 25-383-997) may be... mixture of sodium salts of stearoyl lactylic acids and minor proportions of sodium salts of related acids...

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.

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

Fitting CRISPR-associated Cas3 into the helicase family tree.

PubMed

Jackson, Ryan N; Lavin, Matthew; Carter, Joshua; Wiedenheft, Blake

2014-02-01

Helicases utilize NTPs to modulate their binding to nucleic acids and many of these enzymes also unwind DNA or RNA duplexes in an NTP-dependent fashion. These proteins are phylogenetically related but functionally diverse, with essential roles in virtually all aspects of nucleic acid metabolism. A new class of helicases associated with RNA-guided adaptive immune systems in bacteria and archaea has recently been identified. Prokaryotes acquire resistance to invading genetic parasites by integrating short fragments of foreign nucleic acids into repetitive loci in the host chromosome known as CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPR-associated gene 3 (cas3) encodes a conserved helicase protein that is essential for phage defense. Here we review recent advances in Cas3 biology, and provide a new phylogenetic framework that positions Cas3 in the helicase family tree. We anticipate that this Cas3 phylogeny will guide future biochemical and structural studies. Copyright © 2014. Published by Elsevier Ltd.

21 CFR 184.1260 - Copper gluconate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... (CH2OH(CHOH)4COO)2Cu, CAS Reg. No. 527-09-3) is a substance that occurs as light blue to bluish-green, odorless crystals, or as a fine, light blue powder. It is prepared by the reaction of gluconic acid...

21 CFR 184.1260 - Copper gluconate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... (CH2OH(CHOH)4COO)2Cu, CAS Reg. No. 527-09-3) is a substance that occurs as light blue to bluish-green, odorless crystals, or as a fine, light blue powder. It is prepared by the reaction of gluconic acid...

21 CFR 184.1260 - Copper gluconate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... (CH2OH(CHOH)4COO)2Cu, CAS Reg. No. 527-09-3) is a substance that occurs as light blue to bluish-green, odorless crystals, or as a fine, light blue powder. It is prepared by the reaction of gluconic acid...

21 CFR 184.1260 - Copper gluconate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... (CH2OH(CHOH)4COO)2Cu, CAS Reg. No. 527-09-3) is a substance that occurs as light blue to bluish-green, odorless crystals, or as a fine, light blue powder. It is prepared by the reaction of gluconic acid...

40 CFR 180.930 - Inert ingredients applied to animals; exemptions from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... di- and monohydrogen phosphate esters and the corresponding ammonium, calcium, magnesium, monoethanolamine, potassium, sodium and zinc salts of the phosphate esters; minimum oxyethylene content averages 2..., density control agent Benzoic acid Preservative for formulations 2-Bromo-2-nitro-1,3-propanediol (CAS Reg...

40 CFR 180.930 - Inert ingredients applied to animals; exemptions from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... without polyoxypropylene, mixture of di- and monohydrogen phosphate esters and the corresponding ammonium, calcium, magnesium, monoethanolamine, potassium, sodium and zinc salts of the phosphate esters; minimum..., density control agent Benzoic acid Preservative for formulations 2-Bromo-2-nitro-1,3-propanediol (CAS Reg...

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.

21 CFR 184.1768 - Sodium lactate.

Code of Federal Regulations, 2014 CFR

2014-04-01

....1768 Sodium lactate. (a) Sodium lactate (C3H5O3Na, CAS Reg. No. 72-17-3) is the sodium salt of lactic acid. It is prepared commercially by the neutralization of lactic acid with sodium hydroxide. (b) The... ingredient is used in food at levels not to exceed current good manufacturing practice. (d) Prior sanctions...

40 CFR 180.930 - Inert ingredients applied to animals; exemptions from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

...-hydroxypolyoxyethylene polymer with or without polyoxypropylene, mixture of di- and monohydrogen phosphate esters and the corresponding ammonium, calcium, magnesium, monoethanolamine, potassium, sodium and zinc salts of the phosphate..., density control agent Benzoic acid Preservative for formulations 2-Bromo-2-nitro-1,3-propanediol (CAS Reg...

CatReg Software for Categorical Regression Analysis (Jul 2012)

EPA Science Inventory

CatReg is a computer program, written in the R (http://cran.r-project.org) programming language, to support the conduct of exposure-response analyses by toxicologists and health scientists. CatReg can be used to perform categorical regressi...

CatReg Software for Categorical Regression Analysis (Nov 2006)

EPA Science Inventory

CatReg is a computer program, written in the R (http://cran.r-project.org) programming language, to support the conduct of exposure-response analyses by toxicologists and health scientists. CatReg can be used to perform categorical regressi...

CatReg Software for Categorical Regression Analysis (Feb 2011)

EPA Science Inventory

CatReg is a computer program, written in the R (http://cran.r-project.org) programming language, to support the conduct of exposure-response analyses by toxicologists and health scientists. CatReg can be used to perform categorical regressi...

21 CFR 177.2910 - Ultra-filtration membranes.

Code of Federal Regulations, 2010 CFR

2010-04-01

... of a sintered carbon support that is coated with zirconium oxide (CAS Reg. No. 1314-23-4) containing... of an aluminum oxide support that is coated with zirconium oxide (CAS Reg. No. 1314-23-4) containing...

The Reg1-interacting proteins, Bmh1, Bmh2, Ssb1, and Ssb2, have roles in maintaining glucose repression in Saccharomyces cerevisiae.

PubMed

Dombek, Kenneth M; Kacherovsky, Nataly; Young, Elton T

2004-09-10

In Saccharomyces cerevisiae, a type 1 protein phosphatase complex composed of the Glc7 catalytic subunit and the Reg1 regulatory subunit represses expression of many glucose-regulated genes. Here we show that the Reg1-interacting proteins Bmh1, Bmh2, Ssb1, and Ssb2 have roles in glucose repression. Deleting both BMH genes causes partially constitutive ADH2 expression without significantly increasing the level of Adr1 protein, the major activator of ADH2 expression. Adr1 and Bcy1, the regulatory subunit of cAMP-dependent protein kinase, are both required for this effect indicating that constitutive expression in Deltabmh1Deltabmh2 cells uses the same activation pathway that operates in Deltareg1 cells. Deletion of both BMH genes and REG1 causes a synergistic relief from repression, suggesting that Bmh proteins also act independently of Reg1 during glucose repression. A two-hybrid interaction with the Bmh proteins was mapped to amino acids 187-232, a region of Reg1 that is conserved in different classes of fungi. Deleting this region partially releases SUC2 from glucose repression. This indicates a role for the Reg1-Bmh interaction in glucose repression and also suggests a broad role for Bmh proteins in this process. An in vivo Reg1-Bmh interaction was confirmed by copurification of Bmh proteins with HA(3)-TAP-tagged Reg1. The nonconventional heat shock proteins Ssb1 and Ssb2 are also copurified with HA(3)-TAP-tagged Reg1. Deletion of both SSB genes modestly decreases repression of ADH2 expression in the presence of glucose, suggesting that Ssb proteins, perhaps through their interaction with Reg1, play a minor role in glucose repression.

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.

Sensitivity of simulated South America Climate to the Land Surface Schemes in RegCM4

NASA Astrophysics Data System (ADS)

Llopart, Marta; da Rocha, Rosmeri; Reboita, Michelle; Cuadra, Santiago

2017-04-01

This work evaluates the impact of two land surface parameterizations on the simulated climate and its variability over South America (SA). Two numerical experiments using RegCM4 coupled with Biosphere-Atmosphere Transfer Scheme (RegBATS) and Community Land Model version 3.5 (RegCLM) land surface schemes are compared. For the period 1979-2008, RegCM4 simulations used 50 km horizontal grid spacing and the ERA-Interim reanalysis as initial and boundary conditions. For the period studied, both simulations represent the main observed spatial patterns of rainfall, air temperature and low level circulation over SA. However, concerning the precipitation intensity, RegCLM values are closer to the observations than RegBATS (it is in general, wetter) over most of SA. RegCLM also provides smaller biases for air temperature. Over the Amazon basin, the amplitudes of the annual cycles of the soil moisture, evapotranspiration and sensible heat flux are higher in RegBATS than in RegCLM. This indicates that RegBATS provides large amounts of water vapor to the atmosphere and has more available energy to increase the boundary layer and make it reach the level of free convection (higher sensible heat flux values) resulting in higher precipitation rates and a large wet bias. RegCLM is closer to the observations than RegBATS, presenting smaller wet and warm biases over the Amazon basin. On an interannual scale, the magnitudes of the anomalies of the precipitation and air temperature simulated by RegCLM are closer to the observations. In general, RegBATS simulates higher magnitude for the interannual variability signal.

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.

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

Sensitivity of simulated South America climate to the land surface schemes in RegCM4

NASA Astrophysics Data System (ADS)

Llopart, Marta; da Rocha, Rosmeri P.; Reboita, Michelle; Cuadra, Santiago

2017-12-01

This work evaluates the impact of two land surface parameterizations on the simulated climate and its variability over South America (SA). Two numerical experiments using RegCM4 coupled with the Biosphere-Atmosphere Transfer Scheme (RegBATS) and the Community Land Model version 3.5 (RegCLM) land surface schemes are compared. For the period 1979-2008, RegCM4 simulations used 50 km horizontal grid spacing and the ERA-Interim reanalysis as initial and boundary conditions. For the period studied, both simulations represent the main observed spatial patterns of rainfall, air temperature and low level circulation over SA. However, with regard to the precipitation intensity, RegCLM values are closer to the observations than RegBATS (it is wetter in general) over most of SA. RegCLM also produces smaller biases for air temperature. Over the Amazon basin, the amplitudes of the annual cycles of the soil moisture, evapotranspiration and sensible heat flux are higher in RegBATS than in RegCLM. This indicates that RegBATS provides large amounts of water vapor to the atmosphere and has more available energy to increase the boundary layer thickness and cause it to reach the level of free convection (higher sensible heat flux values) resulting in higher precipitation rates and a large wet bias. RegCLM is closer to the observations than RegBATS, presenting smaller wet and warm biases over the Amazon basin. On an interannual scale, the magnitudes of the anomalies of the precipitation and air temperature simulated by RegCLM are closer to the observations. In general, RegBATS simulates higher magnitude for the interannual variability signal.

21 CFR 74.2705 - FD&C Yellow No. 5.

Code of Federal Regulations, 2010 CFR

2010-04-01

... salt, not more than 0.2 percent. 4,5-Dihydro-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylic acid, disodium salt, not more than 0.2 percent. Ethyl or methyl 4,5-dihydro-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3...-sulfophenyl)azo]-1H-pyrazole-3-carboxylic acid (CAS Reg. No. 1934-21-0). To manufacture the additive, 4...

21 CFR 184.1277 - Dextrin.

Code of Federal Regulations, 2014 CFR

2014-04-01

....1277 Dextrin. (a) Dextrin ((C6H10O5)n·H2O, CAS Reg. No. 9004-53-9) is an incompletely hydrolyzed starch..., or sago starches, or by dry heating the starches after: (1) Treatment with safe and suitable alkalis, acids, or pH control agents and (2) drying the acid or alkali treated starch. (b) The ingredient meets...

21 CFR 184.1625 - Potassium citrate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Potassium citrate. 184.1625 Section 184.1625 Food... GRAS § 184.1625 Potassium citrate. (a) Potassium citrate (C6H5K3O7·H2O, CAS Reg. No. 006100-0905-096) is the potassium salt of citric acid. It is prepared by neutralizing citric acid with potassium...

21 CFR 186.1771 - Sodium palmitate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium palmitate. 186.1771 Section 186.1771 Food... GRAS § 186.1771 Sodium palmitate. (a) Sodium palmitate (C16H31O2Na, CAS Reg. No. 408-35-5) is the sodium salt of palmitic acid (hexadecanoic acid). It exists as a white to yellow powder. Commercially...

21 CFR 186.1771 - Sodium palmitate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium palmitate. 186.1771 Section 186.1771 Food... of Specific Substances Affirmed as GRAS § 186.1771 Sodium palmitate. (a) Sodium palmitate (C16H31O2Na, CAS Reg. No. 408-35-5) is the sodium salt of palmitic acid (hexadecanoic acid). It exists as a white...

21 CFR 186.1771 - Sodium palmitate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium palmitate. 186.1771 Section 186.1771 Food... of Specific Substances Affirmed as GRAS § 186.1771 Sodium palmitate. (a) Sodium palmitate (C16H31O2Na, CAS Reg. No. 408-35-5) is the sodium salt of palmitic acid (hexadecanoic acid). It exists as a white...

21 CFR 186.1771 - Sodium palmitate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium palmitate. 186.1771 Section 186.1771 Food... of Specific Substances Affirmed as GRAS § 186.1771 Sodium palmitate. (a) Sodium palmitate (C16H31O2Na, CAS Reg. No. 408-35-5) is the sodium salt of palmitic acid (hexadecanoic acid). It exists as a white...

21 CFR 186.1771 - Sodium palmitate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium palmitate. 186.1771 Section 186.1771 Food... of Specific Substances Affirmed as GRAS § 186.1771 Sodium palmitate. (a) Sodium palmitate (C16H31O2Na, CAS Reg. No. 408-35-5) is the sodium salt of palmitic acid (hexadecanoic acid). It exists as a white...

21 CFR 184.1199 - Calcium gluconate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium gluconate. 184.1199 Section 184.1199 Food... GRAS § 184.1199 Calcium gluconate. (a) Calcium gluconate ([CH2OH(CHOH)4COO]2Ca, CAS Reg. No. 299-28-5) is the calcium salt of gluconic acid which may be produced by neutralization of gluconic acid with...

Assessment of the simulated climate in two versions of the RegT-Band

NASA Astrophysics Data System (ADS)

da Rocha, Rosmeri; Reboita, Michelle; Llopart, Marta

2017-04-01

This study evaluates two simulations carried out with the tropical band version of the Regional Climate Model (RegT-Band). The purpose was to compare the performance of the RegCM 4.4.5 and 4.6 versions (RegT4.4.5 and RegT4.6). The domain used in the simulations extends from 45° S to 45° N and covers all tropical longitudes, with grid spacing of 39 km, 18 sigma-pressure vertical levels. The initial and boundary conditions for the simulations were provided by ERA-Interim reanalysis and the analyzed period is from January 2005 to December 2008. Regarding the physical parameterizations schemes were used the Emanuel scheme to solve cumulus convection and Community Land Model version 4.5 (CLM4.5) to surface-atmosphere interactions. Seasonal simulated precipitation was compared with Global Precipitation Climatology Project (GPCP) while 2 meters air temperature with ERA-Interim reanalysis. The main results of this study are that RegT4.6 reduces the wet bias over the oceans and the cold bias over the continents compared with RegT4.4.5. In austral summer, RegT4.6 improves the simulation reducing the precipitation amounts mainly over Indian Ocean, Indonesia and eastern northeastern Brazil. However, both versions underestimate the precipitation over the South America Convergence Zone (SACZ). During the austral winter, RegT4.6 simulates the precipitation similar to GPCP over India and it reduces the cold bias over this country compared with RegT4.4.5. However, over the South of Africa, Australia and central-southeast South America, RegT4.6 simulates a strong warm bias.

Cigarette advertising and children's smoking: why Reg was withdrawn.

PubMed Central

Hastings, G. B.; Ryan, H.; Teer, P.; MacKintosh, A. M.

1994-01-01

OBJECTIVE--To examine the appeal of the Embassy Regal "Reg" campaign to young people. DESIGN--Three quantitative surveys and one piece of qualitative research: (a) self completion questionnaire administered in classrooms, (b) questionnaire led interviews with children, (c) questionnaire led interviews with adults, and (d) group discussions with children and adults. SETTINGS--(a) Secondary and middle schools in England; (b) north of England, Scotland, and Wales; (c) north of England, Scotland, and Wales; and (d) Glasgow. SUBJECTS--(a) 5451 schoolchildren aged 11-15 recruited by stratified random sampling; (b) 437 children aged 5-10 recruited by quota sampling; (c) 814 adults aged 15-65 recruited by quota sampling; and (d) 12 groups of children aged 10-15, three groups of adults aged 18-24, and three groups of adults aged 35-55. RESULTS--Children were familiar with cigarette advertising and in particular the Reg campaign. Although younger children struggled to understand the creative content of the adverts, older and smoking children could understand and appreciate the humour. They considered Reg to be amusing and could relate to the type of joke used in the advert. In addition Reg's flippant attitude towards serious issues appealed to the children. While adults aged 18-24 understood the campaign they did not identify with it, and 35-55 year olds (the campaign's supposed target) were unappreciative of the campaign. CONCLUSIONS--The Reg campaign was getting through to children more effectively than it was to adults and held most appeal for teenagers, particularly 14-15 year old smokers. It clearly contravened the code governing tobacco advertising, which states that advertising must not appeal to children more than it does to adults, and it may have had a direct impact on teenage smoking. In view of these findings the Advertising Standards Authority's decision to withdraw the Reg campaign seems appropriate. Images p935-a PMID:7950668

Cigarette advertising and children's smoking: why Reg was withdrawn.

PubMed

Hastings, G B; Ryan, H; Teer, P; MacKintosh, A M

1994-10-08

To examine the appeal of the Embassy Regal "Reg" campaign to young people. Three quantitative surveys and one piece of qualitative research: (a) self completion questionnaire administered in classrooms, (b) questionnaire led interviews with children, (c) questionnaire led interviews with adults, and (d) group discussions with children and adults. (a) Secondary and middle schools in England; (b) north of England, Scotland, and Wales; (c) north of England, Scotland, and Wales; and (d) Glasgow. (a) 5451 schoolchildren aged 11-15 recruited by stratified random sampling; (b) 437 children aged 5-10 recruited by quota sampling; (c) 814 adults aged 15-65 recruited by quota sampling; and (d) 12 groups of children aged 10-15, three groups of adults aged 18-24, and three groups of adults aged 35-55. Children were familiar with cigarette advertising and in particular the Reg campaign. Although younger children struggled to understand the creative content of the adverts, older and smoking children could understand and appreciate the humour. They considered Reg to be amusing and could relate to the type of joke used in the advert. In addition Reg's flippant attitude towards serious issues appealed to the children. While adults aged 18-24 understood the campaign they did not identify with it, and 35-55 year olds (the campaign's supposed target) were unappreciative of the campaign. The Reg campaign was getting through to children more effectively than it was to adults and held most appeal for teenagers, particularly 14-15 year old smokers. It clearly contravened the code governing tobacco advertising, which states that advertising must not appeal to children more than it does to adults, and it may have had a direct impact on teenage smoking. In view of these findings the Advertising Standards Authority's decision to withdraw the Reg campaign seems appropriate.

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

Coupling of Community Land Model with RegCM4 for Indian Summer Monsoon Simulation

NASA Astrophysics Data System (ADS)

Maurya, R. K. S.; Sinha, P.; Mohanty, M. R.; Mohanty, U. C.

2017-11-01

Three land surface schemes available in the regional climate model RegCM4 have been examined to understand the coupling between land and atmosphere for simulation of the Indian summer monsoon rainfall. The RegCM4 is coupled with biosphere-atmosphere transfer scheme (BATS) and the National Center for Atmospheric Research (NCAR) Community Land Model versions 3.5, and 4.5 (CLM3.5 and CLM4.5, respectively) and model performance is evaluated for recent drought (2009) and normal (2011) monsoon years. The CLM4.5 has a more distinct category of surface and it is capable of representing better the land surface characteristics. National Centers for Environmental Prediction (NCEP) and Department of Energy (DOE) reanalysis version 2 (NNRP2) datasets are considered as driving force to conduct the experiments for the Indian monsoon region (30°E-120°E; 30°S-50°N). The NNRP2 and India Meteorological Department (IMD) gridded precipitation data are used for verification analysis. The results indicate that RegCM4 simulations with CLM4.5 (RegCM4-CLM4.5) and CLM3.5 (RegCM4-CLM3.5) surface temperature (at 2 ms) have very low warm biases ( 1 °C), while with BATS (RegCM4-BATS) has a cold bias of about 1-3 °C in peninsular India and some parts of central India. Warm bias in the RegCM4-BATS is observed over the Indo-Gangetic plain and northwest India and the bias is more for the deficit year as compared to the normal year. However, the warm (cold) bias is less in RegCM4-CLM4.5 than other schemes for both the deficit and normal years. The model-simulated maximum (minimum) surface temperature and sensible heat flux at the surface are positively (negatively) biased in all the schemes; however, the bias is higher in RegCM4-BATS and lower in RegCM4-CLM4.5 over India. All the land surface schemes overestimated the precipitation in peninsular India and underestimated in central parts of India for both the years; however, the biases are less in RegCM4-CLM4.5 and more in RegCM4-CLM3.5 and Reg

RIFM fragrance ingredient safety assessment, linalyl benzoate, CAS Registry Number 126-64-7.

PubMed

Api, A M; Belsito, D; Bhatia, S; Bruze, M; Calow, P; Dagli, M L; Dkant, W; Fryer, A D; Kromidas, L; La Cava, S; Lalko, J F; Lapczynski, A; Liebler, D C; Penning, T M; Politano, V T; Ritacco, G; Salvito, D; Schultz, T W; Shen, J; Sipes, I G; Wall, B; Wilcox, D K

2016-11-01

The use of this material under current conditions is supported by existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data show that this material is not genotoxic. Data from the suitable read across analog linalyl phenylacetate (CAS # 7143-69-3) show that this material does not have skin sensitization potential. The repeated dose toxicity endpoint was completed using linalyl cinnamate (CAS # 78-37-5) as a suitable read across analog, which provided a MOE > 100. The developmental and reproductive toxicity endpoint was completed using linalool (CAS # 78-70-6), dehydrolinalool (CAS # 29171-20-8), benzoic acid (CAS # 65-85-0) and sodium benzoate (CAS # 532-32-1) as suitable read across analogs, which provided a MOE > 100. The local respiratory toxicity endpoint was completed using linalool (CAS # 78-70-6) and benzoic acid (CAS # 65-85-0) as suitable read across analogs, which provided a MOE > 100. The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework along with data from the suitable read across analog linalyl cinnamate (CAS # 78-375). Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

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.

HaploReg v4: systematic mining of putative causal variants, cell types, regulators and target genes for human complex traits and disease.

PubMed

Ward, Lucas D; Kellis, Manolis

2016-01-04

More than 90% of common variants associated with complex traits do not affect proteins directly, but instead the circuits that control gene expression. This has increased the urgency of understanding the regulatory genome as a key component for translating genetic results into mechanistic insights and ultimately therapeutics. To address this challenge, we developed HaploReg (http://compbio.mit.edu/HaploReg) to aid the functional dissection of genome-wide association study (GWAS) results, the prediction of putative causal variants in haplotype blocks, the prediction of likely cell types of action, and the prediction of candidate target genes by systematic mining of comparative, epigenomic and regulatory annotations. Since first launching the website in 2011, we have greatly expanded HaploReg, increasing the number of chromatin state maps to 127 reference epigenomes from ENCODE 2012 and Roadmap Epigenomics, incorporating regulator binding data, expanding regulatory motif disruption annotations, and integrating expression quantitative trait locus (eQTL) variants and their tissue-specific target genes from GTEx, Geuvadis, and other recent studies. We present these updates as HaploReg v4, and illustrate a use case of HaploReg for attention deficit hyperactivity disorder (ADHD)-associated SNPs with putative brain regulatory mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

21 CFR 184.1212 - Calcium pantothenate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... ((C9H16NO5)2Ca, CAS Reg. No. of the D-isomer, 137-08-6) is a salt of pantothenic acid, one of the vitamins of the B complex. Only the D-isomer of pantothenic acid has vitamin activity, although both the D-isomer...)(2) of the Act. (d) Prior sanctions for this ingredient different from the uses established in this...

21 CFR 186.1557 - Tall oil.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Tall oil. 186.1557 Section 186.1557 Food and Drugs... Substances Affirmed as GRAS § 186.1557 Tall oil. (a) Tall oil (CAS Reg. No. 8002-26-4) is essentially the sap... consists mainly of tall oil resin acids and tall oil fatty acids. (b) In accordance with § 186.1(b)(1), the...

21 CFR 186.1557 - Tall oil.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Tall oil. 186.1557 Section 186.1557 Food and Drugs... Substances Affirmed as GRAS § 186.1557 Tall oil. (a) Tall oil (CAS Reg. No. 8002-26-4) is essentially the sap... consists mainly of tall oil resin acids and tall oil fatty acids. (b) In accordance with § 186.1(b)(1), the...

21 CFR 186.1557 - Tall oil.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Tall oil. 186.1557 Section 186.1557 Food and Drugs... Substances Affirmed as GRAS § 186.1557 Tall oil. (a) Tall oil (CAS Reg. No. 8002-26-4) is essentially the sap... consists mainly of tall oil resin acids and tall oil fatty acids. (b) In accordance with § 186.1(b)(1), the...

21 CFR 186.1557 - Tall oil.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Tall oil. 186.1557 Section 186.1557 Food and Drugs... Substances Affirmed as GRAS § 186.1557 Tall oil. (a) Tall oil (CAS Reg. No. 8002-26-4) is essentially the sap... consists mainly of tall oil resin acids and tall oil fatty acids. (b) In accordance with § 186.1(b)(1), the...

21 CFR 186.1770 - Sodium oleate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium oleate. 186.1770 Section 186.1770 Food and... Substances Affirmed as GRAS § 186.1770 Sodium oleate. (a) Sodium oleate (C18H33O2Na, CAS Reg. No. 143-19-1) is the sodium salt of oleic acid (cis-9-octadecenoic acid). It exists as a white to yellowish powder...

21 CFR 184.1751 - Sodium citrate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium citrate. 184.1751 Section 184.1751 Food and....1751 Sodium citrate. (a) Sodium citrate (C6H5Na3O7·2H2O, CAS Reg. No. 68-0904-092) is the sodium salt of citric acid. It is prepared by neutralizing citric acid with sodium hydroxide or sodium carbonate...

21 CFR 186.1770 - Sodium oleate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium oleate. 186.1770 Section 186.1770 Food and... Substances Affirmed as GRAS § 186.1770 Sodium oleate. (a) Sodium oleate (C18H33O2Na, CAS Reg. No. 143-19-1) is the sodium salt of oleic acid (cis-9-octadecenoic acid). It exists as a white to yellowish powder...

21 CFR 186.1770 - Sodium oleate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium oleate. 186.1770 Section 186.1770 Food and... Substances Affirmed as GRAS § 186.1770 Sodium oleate. (a) Sodium oleate (C18H33O2Na, CAS Reg. No. 143-19-1) is the sodium salt of oleic acid (cis-9-octadecenoic acid). It exists as a white to yellowish powder...

21 CFR 186.1770 - Sodium oleate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium oleate. 186.1770 Section 186.1770 Food and... Substances Affirmed as GRAS § 186.1770 Sodium oleate. (a) Sodium oleate (C18H33O2Na, CAS Reg. No. 143-19-1) is the sodium salt of oleic acid (cis-9-octadecenoic acid). It exists as a white to yellowish powder...

21 CFR 184.1195 - Calcium citrate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium citrate. 184.1195 Section 184.1195 Food... GRAS § 184.1195 Calcium citrate. (a) Calcium citrate (Ca3(C6H5O7)2·4H2O, CAS Reg. No. 813-0994-095) is the calcium salt of citric acid. It is prepared by neutralizing citric acid with calcium hydroxide or...

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.

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

21 CFR 184.1610 - Potassium alginate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Potassium alginate. 184.1610 Section 184.1610 Food... Specific Substances Affirmed as GRAS § 184.1610 Potassium alginate. (a) Potassium alginate (CAS Reg. No. 9005-36-1) is the potassium salt of alginic acid, a natural polyuronide constituent of certain brown...

21 CFR 184.1634 - Potassium iodide.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Potassium iodide. 184.1634 Section 184.1634 Food... GRAS § 184.1634 Potassium iodide. (a) Potassium iodide (KI, CAS Reg. No. 7681-11-0) is the potassium... reacting hydriodic acid (HI) with potassium bicarbonate (KHCO3). (b) The ingredient meets the...

21 CFR 173.325 - Acidified sodium chlorite solutions.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Acidified sodium chlorite solutions. 173.325... § 173.325 Acidified sodium chlorite solutions. Acidified sodium chlorite solutions may be safely used in... solution of sodium chlorite (CAS Reg. No. 7758-19-2) with any generally recognized as safe (GRAS) acid. (b...

21 CFR 184.1610 - Potassium alginate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Potassium alginate. 184.1610 Section 184.1610 Food... GRAS § 184.1610 Potassium alginate. (a) Potassium alginate (CAS Reg. No. 9005-36-1) is the potassium salt of alginic acid, a natural polyuronide constituent of certain brown algae. Potassium alginate is...

21 CFR 184.1610 - Potassium alginate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Potassium alginate. 184.1610 Section 184.1610 Food... Specific Substances Affirmed as GRAS § 184.1610 Potassium alginate. (a) Potassium alginate (CAS Reg. No. 9005-36-1) is the potassium salt of alginic acid, a natural polyuronide constituent of certain brown...

Stadis{reg_sign} 450 in Merox-sweetened jet fuels

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

Henry, C.P.

1995-05-01

Stadis{reg_sign} 450 has been used in aviation fuels since 1983, and in many cases is the additive of choice due to conductivity retention of treated fuels during distribution, and other characteristics. In the past several years, manufacture of Shell ASA-3 (the other aviation-approved static dissipator additive) has been discontinued; current stores are being drawn down and for some refiners conversion from ASA-3 to Stadis{reg_sign} 450 is underway. In fuels sweetened by hydrogen-treating, Stadis{reg_sign} 450 performs very well and there are few reported difficulties. Chemically sweetened fuels sometimes contain trace materials not removed by the sweetening process. When treated with Stadis{reg_sign}more » 450 some of these fuels have exhibited two behaviors which are being addressed: in one case, the formation of a precipitate which disarmed coalescers; in several other cases, reduced conductivity response and loss of conductivity during storage coupled with unusually large effects on the microseparatometer water separation properties. In late 1992, a Coordinating Research Council (CRC) Panel on Coalescer Deactivation was formed to address these problems. The results of DuPont and CRC efforts are discussed, along with actions taken and underway to eliminate these problems.« less

Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice.

PubMed

Nakagawa, Yoshiko; Sakuma, Tetsushi; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Yanaka, Noriyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

2016-08-15

Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency. © 2016. Published by The Company of Biologists Ltd.

Involvement of the CasK/R two-component system in optimal unsaturation of the Bacillus cereus fatty acids during low-temperature growth.

PubMed

Diomandé, Sara Esther; Nguyen-the, Christophe; Abee, Tjakko; Tempelaars, Marcel H; Broussolle, Véronique; Brillard, Julien

2015-11-20

Bacillus cereus sensu lato is composed of a set of ubiquitous strains including human pathogens that can survive a range of food processing conditions, grow in refrigerated food, and sometimes cause food poisoning. We previously identified the two-component system CasK/R that plays a key role in cold adaptation. To better understand the CasK/R-controlled mechanisms that support low-temperature adaptation, we performed a transcriptomic analysis on the ATCC 14579 strain and its isogenic ∆casK/R mutant grown at 12°C. Several genes involved in fatty acid (FA) metabolism were downregulated in the mutant, including desA and desB encoding FA acyl-lipid desaturases that catalyze the formation of a double-bond on the FA chain in positions ∆5 and ∆10, respectively. A lower proportion of FAs presumably unsaturated by DesA was observed in the ΔcasK/R strain compared to the parental strain while no difference was found for FAs presumably unsaturated by DesB. Addition of phospholipids from egg yolk lecithin rich in unsaturated FAs, to growth medium, abolished the cold-growth impairment of ΔcasK/R suggesting that exogenous unsaturated FAs can support membrane-level modifications and thus compensate for the decreased production of these FAs in the B. cereus ∆casK/R mutant during growth at low temperature. Our findings indicate that CasK/R is involved in the regulation of FA metabolism, and is necessary for cold adaptation of B. cereus unless an exogenous source of unsaturated FAs is available. Copyright © 2015 Elsevier B.V. All rights reserved.

21 CFR 184.1195 - Calcium citrate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium citrate. 184.1195 Section 184.1195 Food and... Substances Affirmed as GRAS § 184.1195 Calcium citrate. (a) Calcium citrate (Ca3(C6H5O7)2·4H2O, CAS Reg. No. 813-0994-095) is the calcium salt of citric acid. It is prepared by neutralizing citric acid with...

21 CFR 184.1751 - Sodium citrate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium citrate. 184.1751 Section 184.1751 Food and... Substances Affirmed as GRAS § 184.1751 Sodium citrate. (a) Sodium citrate (C6H5Na3O7·2H2O, CAS Reg. No. 68-0904-092) is the sodium salt of citric acid. It is prepared by neutralizing citric acid with sodium...

21 CFR 184.1751 - Sodium citrate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium citrate. 184.1751 Section 184.1751 Food and... Substances Affirmed as GRAS § 184.1751 Sodium citrate. (a) Sodium citrate (C6H5Na3O7·2H2O, CAS Reg. No. 68-0904-092) is the sodium salt of citric acid. It is prepared by neutralizing citric acid with sodium...

21 CFR 184.1751 - Sodium citrate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium citrate. 184.1751 Section 184.1751 Food and... Substances Affirmed as GRAS § 184.1751 Sodium citrate. (a) Sodium citrate (C6H5Na3O7·2H2O, CAS Reg. No. 68-0904-092) is the sodium salt of citric acid. It is prepared by neutralizing citric acid with sodium...

21 CFR 184.1751 - Sodium citrate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium citrate. 184.1751 Section 184.1751 Food and... Substances Affirmed as GRAS § 184.1751 Sodium citrate. (a) Sodium citrate (C6H5Na3O7·2H2O, CAS Reg. No. 68-0904-092) is the sodium salt of citric acid. It is prepared by neutralizing citric acid with sodium...

21 CFR 184.1212 - Calcium pantothenate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... ((C9H16NO5)2Ca, CAS Reg. No. of the D-isomer, 137-08-6) is a salt of pantothenic acid, one of the vitamins of the B complex. Only the D-isomer of pantothenic acid has vitamin activity, although both the D-isomer..._regulations/ibr_locations.html. (c) In accordance with § 184.1(b)(1), the ingredient is used in food with no...

21 CFR 184.1212 - Calcium pantothenate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... ((C9H16NO5)2Ca, CAS Reg. No. of the D-isomer, 137-08-6) is a salt of pantothenic acid, one of the vitamins of the B complex. Only the D-isomer of pantothenic acid has vitamin activity, although both the D-isomer..._regulations/ibr_locations.html. (c) In accordance with § 184.1(b)(1), the ingredient is used in food with no...

21 CFR 177.2600 - Rubber articles intended for repeated use.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Rubber articles intended for repeated use. 177... sanction or approval. (3) Substances that by regulation in parts 170 through 189 of this chapter may be... copolymers (CAS Reg. No. 77402-38-1 prepared by reacting a copolymer of omega-laurolactam and adipic acid...

21 CFR 177.2600 - Rubber articles intended for repeated use.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Rubber articles intended for repeated use. 177.2600... packaging. (2) Substances used in accordance with the provisions of a prior sanction or approval. (3... copolymers (CAS Reg. No. 77402-38-1 prepared by reacting a copolymer of omega-laurolactam and adipic acid...

21 CFR 177.2600 - Rubber articles intended for repeated use.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Rubber articles intended for repeated use. 177... sanction or approval. (3) Substances that by regulation in parts 170 through 189 of this chapter may be... copolymers (CAS Reg. No. 77402-38-1 prepared by reacting a copolymer of omega-laurolactam and adipic acid...

21 CFR 184.1643 - Potassium sulfate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Potassium sulfate. 184.1643 Section 184.1643 Food... Specific Substances Affirmed as GRAS § 184.1643 Potassium sulfate. (a) Potassium sulfate (K2SO4, CAS Reg... having a bitter, saline taste. It is prepared by the neutralization of sulfuric acid with potassium...

21 CFR 184.1643 - Potassium sulfate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Potassium sulfate. 184.1643 Section 184.1643 Food... Specific Substances Affirmed as GRAS § 184.1643 Potassium sulfate. (a) Potassium sulfate (K2SO4, CAS Reg... having a bitter, saline taste. It is prepared by the neutralization of sulfuric acid with potassium...

21 CFR 184.1643 - Potassium sulfate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium sulfate. 184.1643 Section 184.1643 Food... Specific Substances Affirmed as GRAS § 184.1643 Potassium sulfate. (a) Potassium sulfate (K2SO4, CAS Reg... having a bitter, saline taste. It is prepared by the neutralization of sulfuric acid with potassium...

21 CFR 184.1724 - Sodium alginate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium alginate. 184.1724 Section 184.1724 Food... Specific Substances Affirmed as GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown algae...

21 CFR 184.1724 - Sodium alginate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium alginate. 184.1724 Section 184.1724 Food... Specific Substances Affirmed as GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown algae...

21 CFR 184.1724 - Sodium alginate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium alginate. 184.1724 Section 184.1724 Food... Specific Substances Affirmed as GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown algae...

21 CFR 184.1643 - Potassium sulfate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Potassium sulfate. 184.1643 Section 184.1643 Food... GRAS § 184.1643 Potassium sulfate. (a) Potassium sulfate (K2SO4, CAS Reg. No. 7778-80-5) occurs.... It is prepared by the neutralization of sulfuric acid with potassium hydroxide or potassium carbonate...

21 CFR 184.1643 - Potassium sulfate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Potassium sulfate. 184.1643 Section 184.1643 Food... Specific Substances Affirmed as GRAS § 184.1643 Potassium sulfate. (a) Potassium sulfate (K2SO4, CAS Reg... having a bitter, saline taste. It is prepared by the neutralization of sulfuric acid with potassium...

21 CFR 184.1187 - Calcium alginate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium alginate. 184.1187 Section 184.1187 Food... GRAS § 184.1187 Calcium alginate. (a) Calcium alginate (CAS Reg. No. 9005-35-0) is the calcium salt of alginic acid, a natural polyuronide constituent of certain brown algae. Calcium alginate is prepared by...

21 CFR 184.1187 - Calcium alginate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium alginate. 184.1187 Section 184.1187 Food... Specific Substances Affirmed as GRAS § 184.1187 Calcium alginate. (a) Calcium alginate (CAS Reg. No. 9005-35-0) is the calcium salt of alginic acid, a natural polyuronide constituent of certain brown algae...

21 CFR 184.1187 - Calcium alginate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium alginate. 184.1187 Section 184.1187 Food... Specific Substances Affirmed as GRAS § 184.1187 Calcium alginate. (a) Calcium alginate (CAS Reg. No. 9005-35-0) is the calcium salt of alginic acid, a natural polyuronide constituent of certain brown algae...

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations.

PubMed

Palermo, Giulia; Miao, Yinglong; Walker, Ross C; Jinek, Martin; McCammon, J Andrew

2017-07-11

CRISPR-Cas9 has become a facile genome editing technology, yet the structural and mechanistic features underlying its function are unclear. Here, we perform extensive molecular simulations in an enhanced sampling regime, using a Gaussian-accelerated molecular dynamics (GaMD) methodology, which probes displacements over hundreds of microseconds to milliseconds, to reveal the conformational dynamics of the endonuclease Cas9 during its activation toward catalysis. We disclose the conformational transition of Cas9 from its apo form to the RNA-bound form, suggesting a mechanism for RNA recruitment in which the domain relocations cause the formation of a positively charged cavity for nucleic acid binding. GaMD also reveals the conformation of a catalytically competent Cas9, which is prone for catalysis and whose experimental characterization is still limited. We show that, upon DNA binding, the conformational dynamics of the HNH domain triggers the formation of the active state, explaining how the HNH domain exerts a conformational control domain over DNA cleavage [Sternberg SH et al. (2015) Nature , 527 , 110-113]. These results provide atomic-level information on the molecular mechanism of CRISPR-Cas9 that will inspire future experimental investigations aimed at fully clarifying the biophysics of this unique genome editing machinery and at developing new tools for nucleic acid manipulation based on CRISPR-Cas9.

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

PubMed Central

Miao, Yinglong; Walker, Ross C.; Jinek, Martin; McCammon, J. Andrew

2017-01-01

CRISPR-Cas9 has become a facile genome editing technology, yet the structural and mechanistic features underlying its function are unclear. Here, we perform extensive molecular simulations in an enhanced sampling regime, using a Gaussian-accelerated molecular dynamics (GaMD) methodology, which probes displacements over hundreds of microseconds to milliseconds, to reveal the conformational dynamics of the endonuclease Cas9 during its activation toward catalysis. We disclose the conformational transition of Cas9 from its apo form to the RNA-bound form, suggesting a mechanism for RNA recruitment in which the domain relocations cause the formation of a positively charged cavity for nucleic acid binding. GaMD also reveals the conformation of a catalytically competent Cas9, which is prone for catalysis and whose experimental characterization is still limited. We show that, upon DNA binding, the conformational dynamics of the HNH domain triggers the formation of the active state, explaining how the HNH domain exerts a conformational control domain over DNA cleavage [Sternberg SH et al. (2015) Nature, 527, 110–113]. These results provide atomic-level information on the molecular mechanism of CRISPR-Cas9 that will inspire future experimental investigations aimed at fully clarifying the biophysics of this unique genome editing machinery and at developing new tools for nucleic acid manipulation based on CRISPR-Cas9. PMID:28652374

The CRISPR-Cas9 system in Neisseria spp.

PubMed Central

2017-01-01

Abstract Bacteria and archaea possess numerous defense systems to combat viral infections and other mobile genetic elements. Uniquely among these, CRISPR-Cas (clustered, regularly interspaced short palindromic repeats-CRISPR associated) provides adaptive genetic interference against foreign nucleic acids. Here we review recent advances on the CRISPR-Cas9 system in Neisseria spp, with a focus on its biological functions in genetic transfer, its mechanistic features that establish new paradigms and its technological applications in eukaryotic genome engineering. PMID:28369433

Exploiting CRISPR/Cas: Interference Mechanisms and Applications

PubMed Central

Richter, Hagen; Randau, Lennart; Plagens, André

2013-01-01

The discovery of biological concepts can often provide a framework for the development of novel molecular tools, which can help us to further understand and manipulate life. One recent example is the elucidation of the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) that protects bacteria and archaea against viruses or conjugative plasmids. The immunity is based on small RNA molecules that are incorporated into versatile multi-domain proteins or protein complexes and specifically target viral nucleic acids via base complementarity. CRISPR/Cas interference machines are utilized to develop novel genome editing tools for different organisms. Here, we will review the latest progress in the elucidation and application of prokaryotic CRISPR/Cas systems and discuss possible future approaches to exploit the potential of these interference machineries. PMID:23857052

Exploiting CRISPR/Cas: interference mechanisms and applications.

PubMed

Richter, Hagen; Randau, Lennart; Plagens, André

2013-07-12

The discovery of biological concepts can often provide a framework for the development of novel molecular tools, which can help us to further understand and manipulate life. One recent example is the elucidation of the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) that protects bacteria and archaea against viruses or conjugative plasmids. The immunity is based on small RNA molecules that are incorporated into versatile multi-domain proteins or protein complexes and specifically target viral nucleic acids via base complementarity. CRISPR/Cas interference machines are utilized to develop novel genome editing tools for different organisms. Here, we will review the latest progress in the elucidation and application of prokaryotic CRISPR/Cas systems and discuss possible future approaches to exploit the potential of these interference machineries.

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.

21 CFR 178.3910 - Surface lubricants used in the manufacture of metallic articles.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Surface lubricants used in the manufacture of...--hydroxypoly (oxyethylene)-poly (oxypropylene) (CAS Reg. No. 9038-95-3) produced by random condensation of a 1... molecular weight of 1000 α-Lauroyl-Ω-hydroxpoly(oxyethylene) (CAS Reg. No. 9004-81-3) having a minimum...

21 CFR 178.3910 - Surface lubricants used in the manufacture of metallic articles.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Surface lubricants used in the manufacture of...--hydroxypoly (oxyethylene)-poly (oxypropylene) (CAS Reg. No. 9038-95-3) produced by random condensation of a 1... molecular weight of 1000 α-Lauroyl-Ω-hydroxpoly(oxyethylene) (CAS Reg. No. 9004-81-3) having a minimum...

21 CFR 178.3910 - Surface lubricants used in the manufacture of metallic articles.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Surface lubricants used in the manufacture of...--hydroxypoly (oxyethylene)-poly (oxypropylene) (CAS Reg. No. 9038-95-3) produced by random condensation of a 1... molecular weight of 1000 α-Lauroyl-Ω-hydroxpoly(oxyethylene) (CAS Reg. No. 9004-81-3) having a minimum...

21 CFR 184.1426 - Magnesium chloride.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Magnesium chloride. 184.1426 Section 184.1426 Food... GRAS § 184.1426 Magnesium chloride. (a) Magnesium chloride (MgC12·6H2O, CAS Reg. No. 7786-30-3) is a... prepared by dissolving magnesium oxide, hydroxide, or carbonate in aqueous hydrochloric acid solution and...

21 CFR 175.125 - Pressure-sensitive adhesives.

Code of Federal Regulations, 2011 CFR

2011-04-01

....125 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...]amino]-2,6-di-tert-butylphenol (CAS Reg. No. 991-84-4) as an antioxidant/stabilizer at a level not to... exceed 5 percent by weight of the pressure-sensitive adhesive. (9) Butanedioic acid, sulfo-1,4-di-(C9-C11...

21 CFR 175.125 - Pressure-sensitive adhesives.

Code of Federal Regulations, 2010 CFR

2010-04-01

....125 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...]amino]-2,6-di-tert-butylphenol (CAS Reg. No. 991-84-4) as an antioxidant/stabilizer at a level not to... exceed 5 percent by weight of the pressure-sensitive adhesive. (9) Butanedioic acid, sulfo-1,4-di-(C9-C11...

21 CFR 184.1634 - Potassium iodide.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Potassium iodide. 184.1634 Section 184.1634 Food... Specific Substances Affirmed as GRAS § 184.1634 Potassium iodide. (a) Potassium iodide (KI, CAS Reg. No. 7681-11-0) is the potassium salt of hydriodic acid. It occurs naturally in sea water and in salt...

21 CFR 184.1634 - Potassium iodide.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Potassium iodide. 184.1634 Section 184.1634 Food... Specific Substances Affirmed as GRAS § 184.1634 Potassium iodide. (a) Potassium iodide (KI, CAS Reg. No. 7681-11-0) is the potassium salt of hydriodic acid. It occurs naturally in sea water and in salt...

21 CFR 184.1634 - Potassium iodide.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Potassium iodide. 184.1634 Section 184.1634 Food... Specific Substances Affirmed as GRAS § 184.1634 Potassium iodide. (a) Potassium iodide (KI, CAS Reg. No. 7681-11-0) is the potassium salt of hydriodic acid. It occurs naturally in sea water and in salt...

21 CFR 172.829 - Neotame.

Code of Federal Regulations, 2014 CFR

2014-04-01

...) Neotame is the chemical N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine-1-methyl ester (CAS Reg. No... acid (N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine), not more than 1.5 percent. (3) Other... neotame is used as a sugar substitute tablet, L-leucine may be used as a lubricant in the manufacture of...

21 CFR 172.829 - Neotame.

Code of Federal Regulations, 2011 CFR

2011-04-01

... percent on a dry basis. (2) Free dipeptide acid (N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine... Multipurpose Additives § 172.829 Neotame. (a) Neotame is the chemical N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine-1-methyl ester (CAS Reg. No. 165450-17-9). (b) Neotame meets the following specifications when it...

21 CFR 172.829 - Neotame.

Code of Federal Regulations, 2013 CFR

2013-04-01

... percent on a dry basis. (2) Free dipeptide acid (N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine... Multipurpose Additives § 172.829 Neotame. (a) Neotame is the chemical N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine-1-methyl ester (CAS Reg. No. 165450-17-9). (b) Neotame meets the following specifications when it...

21 CFR 172.829 - Neotame.

Code of Federal Regulations, 2010 CFR

2010-04-01

... percent on a dry basis. (2) Free dipeptide acid (N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine... Multipurpose Additives § 172.829 Neotame. (a) Neotame is the chemical N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine-1-methyl ester (CAS Reg. No. 165450-17-9). (b) Neotame meets the following specifications when it...

21 CFR 172.829 - Neotame.

Code of Federal Regulations, 2012 CFR

2012-04-01

... percent on a dry basis. (2) Free dipeptide acid (N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine... Multipurpose Additives § 172.829 Neotame. (a) Neotame is the chemical N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine-1-methyl ester (CAS Reg. No. 165450-17-9). (b) Neotame meets the following specifications when it...

21 CFR 184.1724 - Sodium alginate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium alginate. 184.1724 Section 184.1724 Food... GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown algae. Sodium alginate is prepared by the...

21 CFR 184.1784 - Sodium propionate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium propionate. 184.1784 Section 184.1784 Food... GRAS § 184.1784 Sodium propionate. (a) Sodium propionate (C3H5NaO2, CAS Reg. No. 137-40-6) is the sodium salt of propionic acid. It occurs as colorless, transparent crystals or a granular crystalline...

21 CFR 184.1724 - Sodium alginate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium alginate. 184.1724 Section 184.1724 Food and... Substances Affirmed as GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown algae. Sodium alginate is...

21 CFR 186.1756 - Sodium formate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium formate. 186.1756 Section 186.1756 Food and....1756 Sodium formate. (a) Sodium formate (CHNaO2, CAS Reg. No. 141-53-7) is the sodium salt of formic acid. It is produced by the reaction of carbon monoxide with sodium hydroxide. (b) The ingredient is...

21 CFR 186.1797 - Sodium sulfate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium sulfate. 186.1797 Section 186.1797 Food and....1797 Sodium sulfate. (a) Sodium sulfate (Na2SO4, CAS Reg. No. 7757-82-6), also known as Glauber's salt... by the neutralization of sulfuric acid with sodium hydroxide. (b) The ingredient is used as a...

21 CFR 186.1797 - Sodium sulfate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium sulfate. 186.1797 Section 186.1797 Food and... Substances Affirmed as GRAS § 186.1797 Sodium sulfate. (a) Sodium sulfate (Na2SO4, CAS Reg. No. 7757-82-6... crystalline powder. It is prepared by the neutralization of sulfuric acid with sodium hydroxide. (b) The...

21 CFR 186.1797 - Sodium sulfate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium sulfate. 186.1797 Section 186.1797 Food and... Substances Affirmed as GRAS § 186.1797 Sodium sulfate. (a) Sodium sulfate (Na2SO4, CAS Reg. No. 7757-82-6... crystalline powder. It is prepared by the neutralization of sulfuric acid with sodium hydroxide. (b) The...

21 CFR 186.1797 - Sodium sulfate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium sulfate. 186.1797 Section 186.1797 Food and... Substances Affirmed as GRAS § 186.1797 Sodium sulfate. (a) Sodium sulfate (Na2SO4, CAS Reg. No. 7757-82-6... crystalline powder. It is prepared by the neutralization of sulfuric acid with sodium hydroxide. (b) The...

21 CFR 186.1797 - Sodium sulfate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium sulfate. 186.1797 Section 186.1797 Food and... Substances Affirmed as GRAS § 186.1797 Sodium sulfate. (a) Sodium sulfate (Na2SO4, CAS Reg. No. 7757-82-6... crystalline powder. It is prepared by the neutralization of sulfuric acid with sodium hydroxide. (b) The...

21 CFR 184.1634 - Potassium iodide.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium iodide. 184.1634 Section 184.1634 Food... Specific Substances Affirmed as GRAS § 184.1634 Potassium iodide. (a) Potassium iodide (KI, CAS Reg. No. 7681-11-0) is the potassium salt of hydriodic acid. It occurs naturally in sea water and in salt...

21 CFR 184.1199 - Calcium gluconate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium gluconate. 184.1199 Section 184.1199 Food... Specific Substances Affirmed as GRAS § 184.1199 Calcium gluconate. (a) Calcium gluconate ([CH2OH(CHOH)4COO]2Ca, CAS Reg. No. 299-28-5) is the calcium salt of gluconic acid which may be produced by...

21 CFR 184.1199 - Calcium gluconate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium gluconate. 184.1199 Section 184.1199 Food... Specific Substances Affirmed as GRAS § 184.1199 Calcium gluconate. (a) Calcium gluconate ([CH2OH(CHOH)4COO]2Ca, CAS Reg. No. 299-28-5) is the calcium salt of gluconic acid which may be produced by...

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

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

Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9

USDA-ARS?s Scientific Manuscript database

The CRISPR/Cas9 system is known for its precise and efficient gene-editing of a targeted region in a variety of organisms including plants. We targeted FAD2 gene region to perform CRISPR/Cas9 gene-editing in peanut. The FAD2 gene encodes fatty acid desaturase which catalyzes the conversion of oleic ...

CRISPR/Cas9 Immune System as a Tool for Genome Engineering.

PubMed

Hryhorowicz, Magdalena; Lipiński, Daniel; Zeyland, Joanna; Słomski, Ryszard

2017-06-01

CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) adaptive immune systems constitute a bacterial defence against invading nucleic acids derived from bacteriophages or plasmids. This prokaryotic system was adapted in molecular biology and became one of the most powerful and versatile platforms for genome engineering. CRISPR/Cas9 is a simple and rapid tool which enables the efficient modification of endogenous genes in various species and cell types. Moreover, a modified version of the CRISPR/Cas9 system with transcriptional repressors or activators allows robust transcription repression or activation of target genes. The simplicity of CRISPR/Cas9 has resulted in the widespread use of this technology in many fields, including basic research, biotechnology and biomedicine.

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.

Versatile Cas9-Driven Subpopulation Selection Toolbox for Lactococcus lactis.

PubMed

van der Els, Simon; James, Jennelle K; Kleerebezem, Michiel; Bron, Peter A

2018-04-15

CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis The Cas9 expression vector pLABTarget, encoding the Streptocccus pyogenes Cas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci. Introduction of a pepN -targeting derivative of pLABTarget into L. lactis strain MG1363 led to a strong reduction in the number of transformants obtained, which did not occur in a pepN deletion derivative of the same strain, demonstrating the specificity and lethality of the Cas9-mediated double-strand breaks in the lactococcal chromosome. Moreover, the same pLABTarget derivative allowed the selection of a pepN deletion subpopulation from its corresponding single-crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives in L. lactis Finally, pLABTarget, which contained sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages, and integrative conjugative elements (ICEs). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting in L. lactis , while the broad-host-range vector used suggests that this toolbox could readily be expanded to other Gram-positive bacteria. IMPORTANCE Mobile genetic elements in Lactococcus lactis and other lactic acid bacteria (LAB) play an important role in dairy fermentation, having both positive and detrimental effects during the production of fermented dairy products. The pLABTarget vector offers an efficient cloning platform for Cas9 application in lactic acid bacteria. Targeting Cas9 toward mobile genetic elements enabled their effective curing, which is of particular interest in the

Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells

PubMed Central

Weiss, Jonathan M.; Bilate, Angelina M.; Gobert, Michael; Ding, Yi; Curotto de Lafaille, Maria A.; Parkhurst, Christopher N.; Xiong, Huizhong; Dolpady, Jayashree; Frey, Alan B.; Ruocco, Maria Grazia; Yang, Yi; Floess, Stefan; Huehn, Jochen; Oh, Soyoung; Li, Ming O.; Niec, Rachel E.; Rudensky, Alexander Y.; Dustin, Michael L.; Littman, Dan R.

2012-01-01

Foxp3 activity is essential for the normal function of the immune system. Two types of regulatory T (T reg) cells express Foxp3, thymus-generated natural T reg (nT reg) cells, and peripherally generated adaptive T reg (iT reg) cells. These cell types have complementary functions. Until now, it has not been possible to distinguish iT reg from nT reg cells in vivo based solely on surface markers. We report here that Neuropilin 1 (Nrp1) is expressed at high levels by most nT reg cells; in contrast, mucosa-generated iT reg and other noninflammatory iT reg cells express low levels of Nrp1. We found that Nrp1 expression is under the control of TGF-β. By tracing nT reg and iT reg cells, we could establish that some tumors have a very large proportion of infiltrating iT reg cells. iT reg cells obtained from highly inflammatory environments, such as the spinal cords of mice with spontaneous autoimmune encephalomyelitis (EAE) and the lungs of mice with chronic asthma, express Nrp1. In the same animals, iT reg cells in secondary lymphoid organs remain Nrp1low. We also determined that, in spontaneous EAE, iT reg cells help to establish a chronic phase of the disease. PMID:22966001

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…

21 CFR 184.1845 - Stannous chloride (anhydrous and dihydrated).

Code of Federal Regulations, 2010 CFR

2010-04-01

.... Anhydrous stannous chloride (SnCl2, CAS Reg. No. 7772-99-8) is the chloride salt of metallic tin. It is prepared by reacting molten tin with either chlorine or gaseous tin tetrachloride. Dihydrated stannous chloride (SnCl2·2H2O, CAS Reg. No. 10025-0969-091) is the chloride salt of metallic tin that contains two...

21 CFR 184.1845 - Stannous chloride (anhydrous and dihydrated).

Code of Federal Regulations, 2013 CFR

2013-04-01

.... Anhydrous stannous chloride (SnCl2, CAS Reg. No. 7772-99-8) is the chloride salt of metallic tin. It is prepared by reacting molten tin with either chlorine or gaseous tin tetrachloride. Dihydrated stannous chloride (SnCl2·2H2O, CAS Reg. No. 10025-69-1) is the chloride salt of metallic tin that contains two...

21 CFR 184.1845 - Stannous chloride (anhydrous and dihydrated).

Code of Federal Regulations, 2011 CFR

2011-04-01

.... Anhydrous stannous chloride (SnCl2, CAS Reg. No. 7772-99-8) is the chloride salt of metallic tin. It is prepared by reacting molten tin with either chlorine or gaseous tin tetrachloride. Dihydrated stannous chloride (SnCl2·2H2O, CAS Reg. No. 10025-0969-091) is the chloride salt of metallic tin that contains two...

21 CFR 184.1845 - Stannous chloride (anhydrous and dihydrated).

Code of Federal Regulations, 2012 CFR

2012-04-01

.... Anhydrous stannous chloride (SnCl2, CAS Reg. No. 7772-99-8) is the chloride salt of metallic tin. It is prepared by reacting molten tin with either chlorine or gaseous tin tetrachloride. Dihydrated stannous chloride (SnCl2·2H2O, CAS Reg. No. 10025-69-1) is the chloride salt of metallic tin that contains two...

21 CFR 184.1193 - Calcium chloride.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium chloride. 184.1193 Section 184.1193 Food... Specific Substances Affirmed as GRAS § 184.1193 Calcium chloride. (a) Calcium chloride (CaCl2·2H2O, CAS Reg. No. 10035-04-8) or anhydrous calcium chloride (CaCl2, CAS Reg. No. 10043-52-4) may be commercially...

21 CFR 184.1193 - Calcium chloride.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium chloride. 184.1193 Section 184.1193 Food... Specific Substances Affirmed as GRAS § 184.1193 Calcium chloride. (a) Calcium chloride (CaCl2·2H2O, CAS Reg. No. 10035-04-8) or anhydrous calcium chloride (CaCl2, CAS Reg. No. 10043-52-4) may be commercially...

regSNPs: a strategy for prioritizing regulatory single nucleotide substitutions

PubMed Central

Teng, Mingxiang; Ichikawa, Shoji; Padgett, Leah R.; Wang, Yadong; Mort, Matthew; Cooper, David N.; Koller, Daniel L.; Foroud, Tatiana; Edenberg, Howard J.; Econs, Michael J.; Liu, Yunlong

2012-01-01

Motivation: One of the fundamental questions in genetics study is to identify functional DNA variants that are responsible to a disease or phenotype of interest. Results from large-scale genetics studies, such as genome-wide association studies (GWAS), and the availability of high-throughput sequencing technologies provide opportunities in identifying causal variants. Despite the technical advances, informatics methodologies need to be developed to prioritize thousands of variants for potential causative effects. Results: We present regSNPs, an informatics strategy that integrates several established bioinformatics tools, for prioritizing regulatory SNPs, i.e. the SNPs in the promoter regions that potentially affect phenotype through changing transcription of downstream genes. Comparing to existing tools, regSNPs has two distinct features. It considers degenerative features of binding motifs by calculating the differences on the binding affinity caused by the candidate variants and integrates potential phenotypic effects of various transcription factors. When tested by using the disease-causing variants documented in the Human Gene Mutation Database, regSNPs showed mixed performance on various diseases. regSNPs predicted three SNPs that can potentially affect bone density in a region detected in an earlier linkage study. Potential effects of one of the variants were validated using luciferase reporter assay. Contact: yunliu@iupui.edu Supplementary information: Supplementary data are available at Bioinformatics online PMID:22611130

21 CFR 184.1315 - Ferrous sulfate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Ferrous sulfate. 184.1315 Section 184.1315 Food... Specific Substances Affirmed as GRAS § 184.1315 Ferrous sulfate. (a) Ferrous sulfate heptahydrate (iron (II) sulfate heptahydrate, FeSO4·7H2O, CAS Reg. No. 7782-63-0) is prepared by the action of sulfuric acid on...

21 CFR 184.1315 - Ferrous sulfate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Ferrous sulfate. 184.1315 Section 184.1315 Food... Specific Substances Affirmed as GRAS § 184.1315 Ferrous sulfate. (a) Ferrous sulfate heptahydrate (iron (II) sulfate heptahydrate, FeSO4·7H2O, CAS Reg. No. 7782-63-0) is prepared by the action of sulfuric acid on...

21 CFR 184.1440 - Magnesium stearate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Magnesium stearate. 184.1440 Section 184.1440 Food... Specific Substances Affirmed as GRAS § 184.1440 Magnesium stearate. (a) Magnesium stearate (Mg(C17H34COO)2, CAS Reg. No. 557-04-0) is the magnesium salt of stearic acid. It is produced as a white precipitate by...

21 CFR 184.1440 - Magnesium stearate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Magnesium stearate. 184.1440 Section 184.1440 Food... Specific Substances Affirmed as GRAS § 184.1440 Magnesium stearate. (a) Magnesium stearate (Mg(C17H34COO)2, CAS Reg. No. 557-04-0) is the magnesium salt of stearic acid. It is produced as a white precipitate by...

21 CFR 184.1440 - Magnesium stearate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Magnesium stearate. 184.1440 Section 184.1440 Food... GRAS § 184.1440 Magnesium stearate. (a) Magnesium stearate (Mg(C17H34COO)2, CAS Reg. No. 557-04-0) is the magnesium salt of stearic acid. It is produced as a white precipitate by the addition of an...

21 CFR 184.1221 - Calcium propionate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Calcium propionate. 184.1221 Section 184.1221 Food... Specific Substances Affirmed as GRAS § 184.1221 Calcium propionate. (a) Calcium propionate (C6H10CaO4, CAS Reg. No. 4075-81-4) is the calcium salt of propionic acid. It occurs as white crystals or a...

21 CFR 184.1221 - Calcium propionate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium propionate. 184.1221 Section 184.1221 Food... Specific Substances Affirmed as GRAS § 184.1221 Calcium propionate. (a) Calcium propionate (C6H10CaO4, CAS Reg. No. 4075-81-4) is the calcium salt of propionic acid. It occurs as white crystals or a...

21 CFR 184.1318 - Glucono delta-lactone.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Glucono delta-lactone. 184.1318 Section 184.1318... Listing of Specific Substances Affirmed as GRAS § 184.1318 Glucono delta-lactone. (a) Glucono delta-lactone (C6H10O6, CAS Reg. No. 90-80-2), also called D-gluconic acid delta-lactone or D-glucono-1,5...

21 CFR 184.1318 - Glucono delta-lactone.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Glucono delta-lactone. 184.1318 Section 184.1318... Listing of Specific Substances Affirmed as GRAS § 184.1318 Glucono delta-lactone. (a) Glucono delta-lactone (C6H10O6, CAS Reg. No. 90-80-2), also called D-gluconic acid delta-lactone or D-glucono-1,5...

21 CFR 184.1366 - Hydrogen peroxide.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Hydrogen peroxide. 184.1366 Section 184.1366 Food... GRAS § 184.1366 Hydrogen peroxide. (a) Hydrogen peroxide (H2O2, CAS Reg. No. 7722-84-1) is also referred to as hydrogen dioxide. It is made by the electrolytic oxidation of sulfuric acid or a sulfate to...

21 CFR 184.1784 - Sodium propionate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium propionate. 184.1784 Section 184.1784 Food... Specific Substances Affirmed as GRAS § 184.1784 Sodium propionate. (a) Sodium propionate (C3H5NaO2, CAS Reg. No. 137-40-6) is the sodium salt of propionic acid. It occurs as colorless, transparent crystals or a...

21 CFR 184.1784 - Sodium propionate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium propionate. 184.1784 Section 184.1784 Food... Specific Substances Affirmed as GRAS § 184.1784 Sodium propionate. (a) Sodium propionate (C3H5NaO2, CAS Reg. No. 137-40-6) is the sodium salt of propionic acid. It occurs as colorless, transparent crystals or a...

21 CFR 184.1784 - Sodium propionate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium propionate. 184.1784 Section 184.1784 Food... Specific Substances Affirmed as GRAS § 184.1784 Sodium propionate. (a) Sodium propionate (C3H5NaO2, CAS Reg. No. 137-40-6) is the sodium salt of propionic acid. It occurs as colorless, transparent crystals or a...

21 CFR 186.1756 - Sodium formate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium formate. 186.1756 Section 186.1756 Food and... Substances Affirmed as GRAS § 186.1756 Sodium formate. (a) Sodium formate (CHNaO2, CAS Reg. No. 141-53-7) is the sodium salt of formic acid. It is produced by the reaction of carbon monoxide with sodium...

21 CFR 184.1784 - Sodium propionate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium propionate. 184.1784 Section 184.1784 Food... Specific Substances Affirmed as GRAS § 184.1784 Sodium propionate. (a) Sodium propionate (C3H5NaO2, CAS Reg. No. 137-40-6) is the sodium salt of propionic acid. It occurs as colorless, transparent crystals or a...

21 CFR 186.1770 - Sodium oleate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium oleate. 186.1770 Section 186.1770 Food and....1770 Sodium oleate. (a) Sodium oleate (C18H33O2Na, CAS Reg. No. 143-19-1) is the sodium salt of oleic.... Commercially, sodium oleate is made by mixing and heating flaked sodium hydroxide and oleic acid. (b) In...

21 CFR 186.1756 - Sodium formate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium formate. 186.1756 Section 186.1756 Food and... Substances Affirmed as GRAS § 186.1756 Sodium formate. (a) Sodium formate (CHNaO2, CAS Reg. No. 141-53-7) is the sodium salt of formic acid. It is produced by the reaction of carbon monoxide with sodium...

21 CFR 186.1756 - Sodium formate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium formate. 186.1756 Section 186.1756 Food and... Substances Affirmed as GRAS § 186.1756 Sodium formate. (a) Sodium formate (CHNaO2, CAS Reg. No. 141-53-7) is the sodium salt of formic acid. It is produced by the reaction of carbon monoxide with sodium...

21 CFR 186.1756 - Sodium formate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium formate. 186.1756 Section 186.1756 Food and... Substances Affirmed as GRAS § 186.1756 Sodium formate. (a) Sodium formate (CHNaO2, CAS Reg. No. 141-53-7) is the sodium salt of formic acid. It is produced by the reaction of carbon monoxide with sodium...

21 CFR 184.1221 - Calcium propionate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium propionate. 184.1221 Section 184.1221 Food... GRAS § 184.1221 Calcium propionate. (a) Calcium propionate (C6H10CaO4, CAS Reg. No. 4075-81-4) is the calcium salt of propionic acid. It occurs as white crystals or a crystalline solid, possessing not more...

21 CFR 184.1229 - Calcium stearate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium stearate. 184.1229 Section 184.1229 Food... GRAS § 184.1229 Calcium stearate. (a) Calcium stearate (Ca(C17H35COO)2, CAS Reg. No. 1529-23-0) is the calcium salt of stearic acid derived from edible sources. It is prepared as a white precipitate by mixing...

21 CFR 184.1221 - Calcium propionate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium propionate. 184.1221 Section 184.1221 Food... Specific Substances Affirmed as GRAS § 184.1221 Calcium propionate. (a) Calcium propionate (C6H10CaO4, CAS Reg. No. 4075-81-4) is the calcium salt of propionic acid. It occurs as white crystals or a...

21 CFR 184.1221 - Calcium propionate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium propionate. 184.1221 Section 184.1221 Food... Specific Substances Affirmed as GRAS § 184.1221 Calcium propionate. (a) Calcium propionate (C6H10CaO4, CAS Reg. No. 4075-81-4) is the calcium salt of propionic acid. It occurs as white crystals or a...

21 CFR 184.1229 - Calcium stearate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium stearate. 184.1229 Section 184.1229 Food... Specific Substances Affirmed as GRAS § 184.1229 Calcium stearate. (a) Calcium stearate (Ca(C17H35COO)2, CAS Reg. No. 1529-23-0) is the calcium salt of stearic acid derived from edible sources. It is prepared as...

21 CFR 184.1229 - Calcium stearate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium stearate. 184.1229 Section 184.1229 Food... Specific Substances Affirmed as GRAS § 184.1229 Calcium stearate. (a) Calcium stearate (Ca(C17H35COO)2, CAS Reg. No. 1529-23-0) is the calcium salt of stearic acid derived from edible sources. It is prepared as...

Reg proteins and their roles in inflammation and cancer of the human digestive system.

PubMed

Zhao, Jie; Wang, Jingyu; Wang, Hao; Lai, Maode

2013-01-01

The regenerating gene (Reg) family is a group of small molecules that includes four members found in various species, although only three are found in human tissues. Their expression is stimulated by certain growth factors or cytokines. The Reg family plays different roles in proliferation, migration, and anti-apoptosis through activating different signaling pathways. Their dysexpression is closely associated with a number of human conditions and diseases such as inflammation and cancer, especially in the human digestive system. Clinically, upregulation of Reg proteins is usually demonstrated in histological sections and sera from cancer patients. Therefore, Reg proteins can predict the progression and prognosis of cancers, especially those of the digestive tract, and can also act as diagnostic markers and therapeutic targets.

75 FR 66429 - Proposed Collection; Comment Request for Regulation Project REG-104924-98

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-28

... DEPARTMENT OF THE TREASURY Internal Revenue Service [REG-104924-98] Proposed Collection; Comment Request for Regulation Project REG- 104924-98 AGENCY: Internal Revenue Service (IRS), Treasury. ACTION... Christophe, (202) 622-3179, or at Internal Revenue Service, Room 6129, 1111 Constitution Avenue, NW...

Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis.

PubMed

Wakefield, Noelle; Rajan, Rakhi; Sontheimer, Erik J

2015-10-07

In many bacteria and archaea, an adaptive immune system (CRISPR-Cas) provides immunity against foreign genetic elements. This system uses CRISPR RNAs (crRNAs) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. Here we study the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR-Cas system from Staphylococcus epidermidis RP62a, a model for Type III-A CRISPR-Cas systems, and define substrate requirements for SeCas6 activity. We find that SeCas6 is necessary and sufficient for full-length crRNA biogenesis in vitro, and that it relies on both sequence and stem-loop structure in the 3' half of the CRISPR repeat for recognition and processing. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

RNA virus interference via CRISPR/Cas13a system in plants.

PubMed

Aman, Rashid; Ali, Zahir; Butt, Haroon; Mahas, Ahmed; Aljedaani, Fatimah; Khan, Muhammad Zuhaib; Ding, Shouwei; Mahfouz, Magdy

2018-01-04

CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.

21 CFR 184.1193 - Calcium chloride.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium chloride. 184.1193 Section 184.1193 Food... GRAS § 184.1193 Calcium chloride. (a) Calcium chloride (CaCl2·2H2O, CAS Reg. No. 10035-04-8) or anhydrous calcium chloride (CaCl2, CAS Reg. No. 10043-52-4) may be commercially obtained as a byproduct in...

CRISPR-Cas Targeting of Host Genes as an Antiviral Strategy.

PubMed

Chen, Shuliang; Yu, Xiao; Guo, Deyin

2018-01-16

Currently, a new gene editing tool-the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) system-is becoming a promising approach for genetic manipulation at the genomic level. This simple method, originating from the adaptive immune defense system in prokaryotes, has been developed and applied to antiviral research in humans. Based on the characteristics of virus-host interactions and the basic rules of nucleic acid cleavage or gene activation of the CRISPR-Cas system, it can be used to target both the virus genome and host factors to clear viral reservoirs and prohibit virus infection or replication. Here, we summarize recent progress of the CRISPR-Cas technology in editing host genes as an antiviral strategy.

Unravelling the structural and mechanistic basis of CRISPR–Cas systems

PubMed Central

van der Oost, John; Westra, Edze R.; Jackson, Ryan N.; Wiedenheft, Blake

2014-01-01

Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR–Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea. PMID:24909109

Unravelling the structural and mechanistic basis of CRISPR-Cas systems.

PubMed

van der Oost, John; Westra, Edze R; Jackson, Ryan N; Wiedenheft, Blake

2014-07-01

Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR-Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea.

CRISPR-Cas Targeting of Host Genes as an Antiviral Strategy

PubMed Central

Chen, Shuliang; Yu, Xiao; Guo, Deyin

2018-01-01

Currently, a new gene editing tool—the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) system—is becoming a promising approach for genetic manipulation at the genomic level. This simple method, originating from the adaptive immune defense system in prokaryotes, has been developed and applied to antiviral research in humans. Based on the characteristics of virus-host interactions and the basic rules of nucleic acid cleavage or gene activation of the CRISPR-Cas system, it can be used to target both the virus genome and host factors to clear viral reservoirs and prohibit virus infection or replication. Here, we summarize recent progress of the CRISPR-Cas technology in editing host genes as an antiviral strategy. PMID:29337866

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.

REG1B as a predictor of childhood stunting in Bangladesh and Peru123

PubMed Central

Peterson, Kristine M; Buss, Janice; Easley, Rebecca; Yang, Zhengyu; Korpe, Poonum S; Niu, Feiyang; Ma, Jennie Z; Olortegui, Maribel Paredes; Haque, Rashidul; Kosek, Margaret N; Petri, William A

2013-01-01

Background: Undernutrition remains a significant problem worldwide, with environmental enteropathy implicated as a contributing factor. An understanding of the pathogenesis and identification of children at risk are critical to the design of more-effective interventions. Objective: The stool regenerating gene 1β (REG1B) protein, which is a putative measure of intestinal injury and repair, was tested as a noninvasive biomarker of future childhood stunting. Design: A total of 222 children from Bangladesh and 97 children from Peru, who were from impoverished communities, were followed from birth through 24 mo of age with anthropometric measures obtained every 3 mo. Stool REG1B protein concentrations were obtained by using an REG1B polyclonal-polyclonal ELISA at 3 mo of age. We tested for the ability of REG1B to forecast future anthropometric shortfalls, independent of known predictors of undernutrition of family income and baseline height and weight. Results: In the Bangladesh cohort of 222 children, higher REG1B concentrations at month 3 were significantly and independently associated with a growth shortfall in a linear regression analysis at months 9, 12, 18, 21, and 24 and, in the Peru cohort, at months 12, 15, 18, 21, and 24. With the use of a mixed model for repeated measurements, higher stool REG1B concentrations at 3 mo were also independently predictive of a lower future length-for-age z score through 24 mo of age (Bangladesh P = 0.006; Peru P = 0.058). Conclusion: The ability of fecal REG1B to predict growth shortfall in independent cohorts of impoverished children from the developing world offers promise as a malnutrition biomarker and supports a role for environmental enteropathy in the pathogenesis of growth shortfall. PMID:23553156

Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch.

PubMed

Hirosawa, Moe; Fujita, Yoshihiko; Parr, Callum J C; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut; Saito, Hirohide

2017-07-27

The CRISPR-Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR-Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Studies in Rheoencephalography (REG)

DTIC Science & Technology

2010-04-01

r o t i d F l o w m L / m i n i R E G A r b . u n i t E K G m V S A P m m H g S h e d b l o o d m l Bodo: Studies in Rheoencephalography (REG). J...Bodo M, Pearce F, Garcia A, Van Albert S, Settle T , Szebe- ni J, Baranyi L, Hartings J, Armonda R . In vivo cerebral blood flow autoregulation...Microsoft, Redmond, WA) spreadsheet for further processing unless published differently. In most cases, the Student t -test was used for statistical

21 CFR 184.1195 - Calcium citrate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Calcium citrate. 184.1195 Section 184.1195 Food... Specific Substances Affirmed as GRAS § 184.1195 Calcium citrate. (a) Calcium citrate (Ca3(C6H5O7)2·4H2O, CAS Reg. No. 813-0994-095) is the calcium salt of citric acid. It is prepared by neutralizing citric...

21 CFR 184.1804 - Sodium potassium tartrate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium potassium tartrate. 184.1804 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1804 Sodium potassium tartrate. (a) Sodium potassium tartrate (C4H4KNaO6·4H2O, CAS Reg. No. 304-59-6) is the sodium potassium salt of l−(+)−tartaric acid and is...

21 CFR 184.1625 - Potassium citrate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Potassium citrate. 184.1625 Section 184.1625 Food... Specific Substances Affirmed as GRAS § 184.1625 Potassium citrate. (a) Potassium citrate (C6H5K3O7·H2O, CAS Reg. No. 006100-0905-096) is the potassium salt of citric acid. It is prepared by neutralizing citric...

21 CFR 184.1804 - Sodium potassium tartrate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium potassium tartrate. 184.1804 Section 184... as GRAS § 184.1804 Sodium potassium tartrate. (a) Sodium potassium tartrate (C4H4KNaO6·4H2O, CAS Reg. No. 304-59-6) is the sodium potassium salt of l−(+)−tartaric acid and is also called the Rochelle...

21 CFR 184.1804 - Sodium potassium tartrate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium potassium tartrate. 184.1804 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1804 Sodium potassium tartrate. (a) Sodium potassium tartrate (C4H4KNaO6·4H2O, CAS Reg. No. 304-59-6) is the sodium potassium salt of l−(+)−tartaric acid and is...

21 CFR 184.1804 - Sodium potassium tartrate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium potassium tartrate. 184.1804 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1804 Sodium potassium tartrate. (a) Sodium potassium tartrate (C4H4KNaO6·4H2O, CAS Reg. No. 304-59-6) is the sodium potassium salt of l−(+)−tartaric acid and is...

21 CFR 184.1625 - Potassium citrate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Potassium citrate. 184.1625 Section 184.1625 Food... Specific Substances Affirmed as GRAS § 184.1625 Potassium citrate. (a) Potassium citrate (C6H5K3O7·H2O, CAS Reg. No. 006100-0905-096) is the potassium salt of citric acid. It is prepared by neutralizing citric...

21 CFR 184.1625 - Potassium citrate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Potassium citrate. 184.1625 Section 184.1625 Food... Specific Substances Affirmed as GRAS § 184.1625 Potassium citrate. (a) Potassium citrate (C6H5K3O7·H2O, CAS Reg. No. 006100-0905-096) is the potassium salt of citric acid. It is prepared by neutralizing citric...

21 CFR 184.1754 - Sodium diacetate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium diacetate. 184.1754 Section 184.1754 Food... Specific Substances Affirmed as GRAS § 184.1754 Sodium diacetate. (a) Sodium diacetate (C4H7O4Na·xH2O, CAS Reg. No. 126-96-5) is a molecular compound of acetic acid, sodium acetate, and water of hydration. The...

21 CFR 184.1754 - Sodium diacetate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium diacetate. 184.1754 Section 184.1754 Food... Specific Substances Affirmed as GRAS § 184.1754 Sodium diacetate. (a) Sodium diacetate (C4H7O4Na·xH2O, CAS Reg. No. 126-96-5) is a molecular compound of acetic acid, sodium acetate, and water of hydration. The...

21 CFR 184.1754 - Sodium diacetate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium diacetate. 184.1754 Section 184.1754 Food... Specific Substances Affirmed as GRAS § 184.1754 Sodium diacetate. (a) Sodium diacetate (C4H7O4Na·xH2O, CAS Reg. No. 126-96-5) is a molecular compound of acetic acid, sodium acetate, and water of hydration. The...

21 CFR 184.1754 - Sodium diacetate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium diacetate. 184.1754 Section 184.1754 Food... Specific Substances Affirmed as GRAS § 184.1754 Sodium diacetate. (a) Sodium diacetate (C4H7O4Na·xH2O, CAS Reg. No. 126-96-5) is a molecular compound of acetic acid, sodium acetate, and water of hydration. The...

21 CFR 184.1754 - Sodium diacetate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium diacetate. 184.1754 Section 184.1754 Food... GRAS § 184.1754 Sodium diacetate. (a) Sodium diacetate (C4H7O4Na·xH2O, CAS Reg. No. 126-96-5) is a molecular compound of acetic acid, sodium acetate, and water of hydration. The technical grade is prepared...

21 CFR 184.1625 - Potassium citrate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium citrate. 184.1625 Section 184.1625 Food... Specific Substances Affirmed as GRAS § 184.1625 Potassium citrate. (a) Potassium citrate (C6H5K3O7·H2O, CAS Reg. No. 006100-0905-096) is the potassium salt of citric acid. It is prepared by neutralizing citric...

21 CFR 184.1185 - Calcium acetate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium acetate. 184.1185 Section 184.1185 Food... Specific Substances Affirmed as GRAS § 184.1185 Calcium acetate. (a) Calcium acetate (Ca (C2H3O2)2, CAS Reg. No. 62-54-4), also known as acetate of lime or vinegar salts, is the calcium salt of acetic acid. It...

21 CFR 184.1185 - Calcium acetate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium acetate. 184.1185 Section 184.1185 Food... GRAS § 184.1185 Calcium acetate. (a) Calcium acetate (Ca (C2H3O2)2, CAS Reg. No. 62-54-4), also known as acetate of lime or vinegar salts, is the calcium salt of acetic acid. It may be produced by the...

21 CFR 184.1185 - Calcium acetate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium acetate. 184.1185 Section 184.1185 Food... Specific Substances Affirmed as GRAS § 184.1185 Calcium acetate. (a) Calcium acetate (Ca (C2H3O2)2, CAS Reg. No. 62-54-4), also known as acetate of lime or vinegar salts, is the calcium salt of acetic acid. It...

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

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.

Reg3β is associated with cardiac inflammation and provides prognostic information in patients with acute coronary syndrome.

PubMed

Lörchner, Holger; Widera, Christian; Hou, Yunlong; Elsässer, Albrecht; Warnecke, Henning; Giannitsis, Evangelos; Hulot, Jean-Sebastien; Braun, Thomas; Wollert, Kai C; Pöling, Jochen

2018-05-01

Regenerating islet-derived protein 3 beta (Reg3β) is a cardiomyocyte-derived chemokine for macrophages that is upregulated after myocardial infarction (MI) in mice. Here, we hypothesized that monitoring Reg3β expression might provide specific information on the degree of cardiac inflammation, which is a key determinant in disease progression and prognosis of patients with acute coronary syndrome (ACS). The expression of Reg3β and other inflammatory markers including C-reactive protein (CRP) and myeloperoxidase (MPO) was measured by immunoblotting at serial time points in the hearts and serum of mice with acute MI. We identified a rapid increase of Reg3β, CRP and MPO expression in cardiac tissue and serum within the first 24 h after MI. The expression of Reg3β peaked at day 4 and thereby paralleled the kinetic profile of the early immune-inflammatory response at sites of cardiac injury, which has been characterized by multicolor flow cytometry. In a retrospective analysis including 322 ACS patients and 117 apparently healthy individuals, we detected increased Reg3β serum concentrations in ACS patients on admission by ELISA. Multiple regression analysis revealed significant relationships between Reg3β and hs-CRP, age, diabetes and NT-proBNP in ACS. Moreover, elevated Reg3β levels on admission were associated with an increased risk of death independent of cardiovascular risk factors and hs-CRP. Reg3β is a prognostic biomarker for ACS and is strongly associated with the intensity of cardiac inflammation. Accordingly, Reg3β may complement established strategies of acute risk assessment in the management of ACS. Copyright © 2018 Elsevier B.V. All rights reserved.

Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas13a enzyme

PubMed Central

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.; Holton, James M.; Charles, Emeric; O’Connell, Mitchell R.; Doudna, Jennifer A.

2018-01-01

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR-Cas systems include the Cas13a enzyme, an RNA-activated ribonuclease (RNase) capable of crRNA processing and single-stranded RNA degradation upon target transcript binding. Here we present the 2.0 Å resolution crystal structure of a crRNA-bound L. bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define for the first time the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked prior to target RNA recognition, with implications for both bacterial immunity and diagnostic applications. PMID:28892041

Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

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

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

DOE PAGES

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.; ...

2017-09-11

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

Gut REG3γ-Associated Lactobacillus Induces Anti-inflammatory Macrophages to Maintain Adipose Tissue Homeostasis

PubMed Central

Huang, Yugang; Qi, HouBao; Zhang, Zhiqian; Wang, Enlin; Yun, Huan; Yan, Hui; Su, Xiaomin; Liu, Yingquan; Tang, Zenzen; Gao, Yunhuan; Shang, Wencong; Zhou, Jiang; Wang, Tianze; Che, Yongzhe; Zhang, Yuan; Yang, Rongcun

2017-01-01

Gut microbiota may not only affect composition of local immune cells but also affect systemic immune cells. However, it is not completely clear how gut microbiota modulate these immune systems. Here, we found that there exist expanded macrophage pools in huREG3γtgIEC mice. REG3γ-associated Lactobacillus, which is homology to Lactobacillus Taiwanese, could enlarge macrophage pools not only in the small intestinal lamina propria but also in the spleen and adipose tissues. STAT3-mediated signal(s) was a critical factor in the Lactobacillus-mediated anti-inflammatory macrophages. We also offered evidence for critical cellular links among REG3γ-associated Lactobacillus, tissue macrophages, and obesity diseases. Anti-inflammatory macrophages in the lamina propria, which are induced by REG3γ-associated Lactobacillus, may migrate into adipose tissues and are involved in resistance against high-fat diet-mediated obesity. Thus, REG3γ-associated Lactobacillus-induced anti-inflammatory macrophages in gut tissues may play a role in adipose tissue homeostasis. PMID:28928739

Expression and Significance of the HIP/PAP and RegIIIγ Antimicrobial Peptides during Mammalian Urinary Tract Infection.

PubMed

Spencer, John David; Jackson, Ashley R; Li, Birong; Ching, Christina B; Vonau, Martin; Easterling, Robert S; Schwaderer, Andrew L; McHugh, Kirk M; Becknell, Brian

2015-01-01

Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified Regenerating islet-derived 3 gamma (RegIIIγ) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic Escherichia coli (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC in vitro, but does kill Staphylococcus saprophyticus in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain.

Expression and Significance of the HIP/PAP and RegIIIγ Antimicrobial Peptides during Mammalian Urinary Tract Infection

PubMed Central

Spencer, John David; Jackson, Ashley R.; Li, Birong; Ching, Christina B.; Vonau, Martin; Easterling, Robert S.; Schwaderer, Andrew L.; McHugh, Kirk M.; Becknell, Brian

2015-01-01

Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified Regenerating islet-derived 3 gamma (RegIIIγ) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic Escherichia coli (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC in vitro, but does kill Staphylococcus saprophyticus in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain. PMID:26658437

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.

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications.

PubMed

Liu, Chang; Zhang, Li; Liu, Hao; Cheng, Kun

2017-11-28

The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation.

PubMed

Musharova, Olga; Klimuk, Evgeny; Datsenko, Kirill A; Metlitskaya, Anastasia; Logacheva, Maria; Semenova, Ekaterina; Severinov, Konstantin; Savitskaya, Ekaterina

2017-04-07

During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR-Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1-Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription.

PubMed

Zhou, Xin X; Zou, Xinzhi; Chung, Hokyung K; Gao, Yuchen; Liu, Yanxia; Qi, Lei S; Lin, Michael Z

2018-02-16

Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

Elimination of the cryptic plasmid in Leuconostoc citreum by CRISPR/Cas9 system.

PubMed

Jang, Ye-Ji; Seo, Seung-Oh; Kim, Seul-Ah; Li, Ling; Kim, Tae-Jip; Kim, Sun Chang; Jin, Yong-Su; Han, Nam Soo

2017-06-10

Leuconostoc spp. are important lactic acid bacteria for the fermentation of foods. In particular, L. citreum strains isolated from various foods have been used as host strains for genetic and metabolic engineering studies. In order to develop a food-grade genetic engineering system, L. citreum CB2567 was isolated from Kimchi. However, the isolated bacterium contained a cryptic plasmid which was difficult to eliminate. As the existence of the plasmid might hinder strain engineering, we eliminated the plasmid using an RNA-guided DNA endonuclease CRISPR/Cas9 system. We demonstrated that a plasmid-free L. citreum CB2567 host strain could be efficiently constructed through a two-step procedure: 1) transformation of the "killer" plasmid expressing Cas9 endonuclease and a guide RNA (gRNA) targeting for a specific sequence in the cryptic plasmid, and 2) serial subculture without antibiotics for curing the killer plasmid. When the crude extract of L. citreum expressing Cas9 and the guide RNA was incubated with a PCR fragment containing the specific sequence recognized by the guide RNA, the PCR fragment was cleaved. Also, the cryptic plasmid pCB42 was successfully eliminated from the host strain after transforming the plasmid harboring Cas9 and the guide RNA. The Cas9 and gRNA expression plasmid used in this study can be applied for genome engineering purposes by additionally introducing an editing DNA template to repair the double strand DNA breakage caused by Cas9 in the genome of L. citreum. This study demonstrates the feasibility of developing CRISPR/Cas9-based genetic engineering tools to develop a safe host strain and construct food-grade lactic acid bacteria without residual antibiotic markers. Copyright © 2017 Elsevier B.V. All rights reserved.

Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles.

PubMed

Mout, Rubul; Rotello, Vincent M

2017-10-20

In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in an E-tag and usually n = 15 or 20), C-terminus nuclear localizing signal (NLS), and a C-terminus 6xHis-tag. [Cas9En hereafter] To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant Cas9En, and sgRNA). Laboratory-synthesis of gold nanoparticles can take up to a few weeks, but can be synthesized in large batches that can be used for many years without compromising the quality. Cas9En can be cloned from a regular SpCas9 gene (Addgene plasmid id = 47327), and expressed and purified using standard laboratory procedures which are not a part of this protocol. Similarly, sgRNA can be laboratory-synthesized using in vitro transcription from a template gene (Addgene plasmid id = 51765) or can be purchased from various sources. Once these materials are ready, it takes about ~30 min to make the Cas9En-RNP complex and 10 min to make the Cas9En-RNP/nanoparticles nanoassemblies, which are immediately used for delivery (Figure 1). Complete delivery (90-95% cytoplasmic and nuclear delivery) is achieved in less than 3 h. Follow-up editing experiments require additional time based on users' need. Synthesis of arginine functionalized gold nanoparticles (ArgNPs) (Yang et al ., 2011), expression of recombinant Cas9En, and in vitro synthesis of sgRNA is reported elsewhere (Mout et al ., 2017). We report here only the generation of the delivery vehicle i.e. , the fabrication of Cas9En-RNP/ArgNPs nanoassembly.

CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level.

PubMed

Yuen, Garmen; Khan, Fehad J; Gao, Shaojian; Stommel, Jayne M; Batchelor, Eric; Wu, Xiaolin; Luo, Ji

2017-11-16

CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

Striking Plasticity of CRISPR-Cas9 and Key Role of Non-target DNA, as Revealed by Molecular Simulations.

PubMed

Palermo, Giulia; Miao, Yinglong; Walker, Ross C; Jinek, Martin; McCammon, J Andrew

2016-10-26

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system recently emerged as a transformative genome-editing technology that is innovating basic bioscience and applied medicine and biotechnology. The endonuclease Cas9 associates with a guide RNA to match and cleave complementary sequences in double stranded DNA, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Although extensive structural studies are ongoing, the conformational dynamics of Cas9 and its interplay with the nucleic acids during association and DNA cleavage are largely unclear. Here, by employing multi-microsecond time scale molecular dynamics, we reveal the conformational plasticity of Cas9 and identify key determinants that allow its large-scale conformational changes during nucleic acid binding and processing. We show how the "closure" of the protein, which accompanies nucleic acid binding, fundamentally relies on highly coupled and specific motions of the protein domains, collectively initiating the prominent conformational changes needed for nucleic acid association. We further reveal a key role of the non-target DNA during the process of activation of the nuclease HNH domain, showing how the nontarget DNA positioning triggers local conformational changes that favor the formation of a catalytically competent Cas9. Finally, a remarkable conformational plasticity is identified as an intrinsic property of the HNH domain, constituting a necessary element that allows for the HNH repositioning. These novel findings constitute a reference for future experimental studies aimed at a full characterization of the dynamic features of the CRISPR-Cas9 system, and-more importantly-call for novel structure engineering efforts that are of fundamental importance for the rational design of new genome-engineering applications.

Metabolic engineering of Schizosaccharomyces pombe via CRISPR-Cas9 genome editing for lactic acid production from glucose and cellobiose.

PubMed

Ozaki, Aiko; Konishi, Rie; Otomo, Chisako; Kishida, Mayumi; Takayama, Seiya; Matsumoto, Takuya; Tanaka, Tsutomu; Kondo, Akihiko

2017-12-01

Modification of the Schizosaccharomyces pombe genome is often laborious, time consuming due to the lower efficiency of homologous recombination. Here, we constructed metabolically engineered S. pombe strains using a CRISPR-Cas9 system and also demonstrated D-lactic acid (D-LA) production from glucose and cellobiose. Genes encoding two separate pyruvate decarboxylases (PDCs), an L-lactic acid dehydrogenase (L-LDH), and a minor alcohol dehydrogenase (SPBC337.11) were disrupted, thereby attenuating ethanol production. To increase the cellular supply of acetyl-CoA, an important metabolite for growth, we introduced genes encoding bacterial acetylating acetaldehyde dehydrogenase enzymes (Escherichia coli MhpF and EutE). D-LA production by the resulting strain was achieved by expressing a Lactobacillus plantarum gene encoding D-lactate dehydrogenase. The engineered strain efficiently consumed glucose and produced D-LA at 25.2 g/L from 35.5 g/L of consumed glucose with a yield of 0.71 g D-LA / g glucose. We further modified this strain by expressing beta-glucosidase by cell surface display; the resulting strain produced D-LA at 24.4 g/L from 30 g/L of cellobiose in minimal medium, with a yield of 0.68 g D-LA / g glucose. To our knowledge, this study represents the first report of a S. pombe strain that was metabolically engineered using a CRISPR-Cas9 system, and demonstrates the possibility of engineering S. pombe for the production of value-added chemicals.

75 FR 47893 - Proposed Collection; Comment Request for REG-111583-07, (T.D. 9405) (Final)

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-09

...-111583-07, (T.D. 9405) (Final) AGENCY: Internal Revenue Service (IRS), Treasury. ACTION: Notice and... comments concerning temporary and final regulations (REG-111583-07) (T.D. 9405), Employment Tax Adjustments... Adjustments. OMB Number: 1545-2097. Form Number: REG-111583-07 (T.D. 9405) (final). Abstract: This document...

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.

[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.

Springing into Action: Reg2 Negatively Regulates Snf1 Protein Kinase and Facilitates Recovery from Prolonged Glucose Starvation in Saccharomyces cerevisiae.

PubMed

Maziarz, Marcin; Shevade, Aishwarya; Barrett, LaKisha; Kuchin, Sergei

2016-07-01

Glucose is the preferred carbon source for the yeast Saccharomyces cerevisiae Glucose limitation activates Snf1 protein kinase, a key regulator of energy homeostasis that promotes utilization of alternative carbon sources and enforces energy conservation. Snf1 activation requires phosphorylation of its T-loop threonine (Thr210) by upstream kinases. When glucose is abundant, Snf1 is inhibited by Thr210 dephosphorylation. This involves the function of the type 1 protein phosphatase Glc7, which is targeted to Snf1 by a regulatory subunit, Reg1. The reg1 mutation causes increased Snf1 activity and mimics various aspects of glucose limitation, including slower growth. Reg2 is another Glc7 regulatory subunit encoded by a paralogous gene, REG2 Previous evidence indicated that the reg2 mutation exacerbates the Snf1-dependent slow-growth phenotype caused by reg1, suggesting a link between Reg2 and Snf1. Here, we explore this link in more detail and present evidence that Reg2 contributes to Snf1 Thr210 dephosphorylation. Consistent with this role, Reg2 interacts with wild-type Snf1 but not with nonphosphorylatable Snf1-T210A. Reg2 accumulation increases in a Snf1-dependent manner during prolonged glucose deprivation, and glucose-starved cells lacking Reg2 exhibit delayed Snf1 Thr210 dephosphorylation and slower growth recovery upon glucose replenishment. Accordingly, cells lacking Reg2 are outcompeted by wild-type cells in the course of several glucose starvation/replenishment cycles. Collectively, our results support a model in which Reg2-Glc7 contributes to the negative control of Snf1 in response to glucose refeeding after prolonged starvation. The competitive growth advantage provided by Reg2 underscores the evolutionary significance of this paralog for S. cerevisiae The ability of microorganisms to respond to stress is essential for their survival. However, rapid recovery from stress could be equally crucial in competitive environments. Therefore, a wise stress response

Springing into Action: Reg2 Negatively Regulates Snf1 Protein Kinase and Facilitates Recovery from Prolonged Glucose Starvation in Saccharomyces cerevisiae

PubMed Central

Maziarz, Marcin; Shevade, Aishwarya; Barrett, LaKisha

2016-01-01

ABSTRACT Glucose is the preferred carbon source for the yeast Saccharomyces cerevisiae. Glucose limitation activates Snf1 protein kinase, a key regulator of energy homeostasis that promotes utilization of alternative carbon sources and enforces energy conservation. Snf1 activation requires phosphorylation of its T-loop threonine (Thr210) by upstream kinases. When glucose is abundant, Snf1 is inhibited by Thr210 dephosphorylation. This involves the function of the type 1 protein phosphatase Glc7, which is targeted to Snf1 by a regulatory subunit, Reg1. The reg1 mutation causes increased Snf1 activity and mimics various aspects of glucose limitation, including slower growth. Reg2 is another Glc7 regulatory subunit encoded by a paralogous gene, REG2. Previous evidence indicated that the reg2 mutation exacerbates the Snf1-dependent slow-growth phenotype caused by reg1, suggesting a link between Reg2 and Snf1. Here, we explore this link in more detail and present evidence that Reg2 contributes to Snf1 Thr210 dephosphorylation. Consistent with this role, Reg2 interacts with wild-type Snf1 but not with nonphosphorylatable Snf1-T210A. Reg2 accumulation increases in a Snf1-dependent manner during prolonged glucose deprivation, and glucose-starved cells lacking Reg2 exhibit delayed Snf1 Thr210 dephosphorylation and slower growth recovery upon glucose replenishment. Accordingly, cells lacking Reg2 are outcompeted by wild-type cells in the course of several glucose starvation/replenishment cycles. Collectively, our results support a model in which Reg2-Glc7 contributes to the negative control of Snf1 in response to glucose refeeding after prolonged starvation. The competitive growth advantage provided by Reg2 underscores the evolutionary significance of this paralog for S. cerevisiae. IMPORTANCE The ability of microorganisms to respond to stress is essential for their survival. However, rapid recovery from stress could be equally crucial in competitive environments. Therefore

Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri.

PubMed

Sanozky-Dawes, Rosemary; Selle, Kurt; O'Flaherty, Sarah; Klaenhammer, Todd; Barrangou, Rodolphe

2015-09-01

Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes. In this study, 17 Lactobacillus gasseri strains were investigated and 6 harboured a type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the protospacer adjacent motif sequence, determined to be 5'-NTAA-3' flanking the 3' end of the protospacer. The system in L. gasseri JV-V03 and NCK 1342 interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native type II-A CRISPR-Cas system in the commensal species L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri, and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.

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

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.

Artificial Virus Delivers CRISPR-Cas9 System for Genome Editing of Cells in Mice.

PubMed

Li, Ling; Song, Linjiang; Liu, Xiaowei; Yang, Xi; Li, Xia; He, Tao; Wang, Ning; Yang, Suleixin; Yu, Chuan; Yin, Tao; Wen, Yanzhu; He, Zhiyao; Wei, Xiawei; Su, Weijun; Wu, Qinjie; Yao, Shaohua; Gong, Changyang; Wei, Yuquan

2017-01-24

CRISPR-Cas9 has emerged as a versatile genome-editing platform. However, due to the large size of the commonly used CRISPR-Cas9 system, its effective delivery has been a challenge and limits its utility for basic research and therapeutic applications. Herein, a multifunctional nucleus-targeting "core-shell" artificial virus (RRPHC) was constructed for the delivery of CRISPR-Cas9 system. The artificial virus could efficiently load with the CRISPR-Cas9 system, accelerate the endosomal escape, and promote the penetration into the nucleus without additional nuclear-localization signal, thus enabling targeted gene disruption. Notably, the artificial virus is more efficient than SuperFect, Lipofectamine 2000, and Lipofectamine 3000. When loaded with a CRISPR-Cas9 plasmid, it induced higher targeted gene disruption efficacy than that of Lipofectamine 3000. Furthermore, the artificial virus effectively targets the ovarian cancer via dual-receptor-mediated endocytosis and had minimum side effects. When loaded with the Cas9-hMTH1 system targeting MTH1 gene, RRPHC showed effective disruption of MTH1 in vivo. This strategy could be adapted for delivering CRISPR-Cas9 plasmid or other functional nucleic acids in vivo.

Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications.

PubMed

Pyzocha, Neena K; Chen, Sidi

2018-02-16

CRISPR-Cas genome editing technologies have revolutionized modern molecular biology by making targeted DNA edits simple and scalable. These technologies are developed by domesticating naturally occurring microbial adaptive immune systems that display wide diversity of functionality for targeted nucleic acid cleavage. Several CRISPR-Cas single effector enzymes have been characterized and engineered for use in mammalian cells. The unique properties of the single effector enzymes can make a critical difference in experimental use or targeting specificity. This review describes known single effector enzymes and discusses their use in genome engineering applications.

Resetting microbiota by Lactobacillus reuteri inhibits T reg deficiency–induced autoimmunity via adenosine A2A receptors

PubMed Central

Hoang, Thomas K.; Tian, Xiangjun; Luo, Meng; Zhou, Jain; Tatevian, Nina; Molina, Jose G.; Blackburn, Michael R.; Gomez, Thomas H.

2017-01-01

Regulatory T (T reg) cell deficiency causes lethal, CD4+ T cell–driven autoimmune diseases. Stem cell transplantation is used to treat these diseases, but this procedure is limited by the availability of a suitable donor. The intestinal microbiota drives host immune homeostasis by regulating the differentiation and expansion of T reg, Th1, and Th2 cells. It is currently unclear if T reg cell deficiency–mediated autoimmune disorders can be treated by targeting the enteric microbiota. Here, we demonstrate that Foxp3+ T reg cell deficiency results in gut microbial dysbiosis and autoimmunity over the lifespan of scurfy (SF) mouse. Remodeling microbiota with Lactobacillus reuteri prolonged survival and reduced multiorgan inflammation in SF mice. L. reuteri changed the metabolomic profile disrupted by T reg cell deficiency, and a major effect was to restore levels of the purine metabolite inosine. Feeding inosine itself prolonged life and inhibited multiorgan inflammation by reducing Th1/Th2 cells and their associated cytokines. Mechanistically, the inhibition of inosine on the differentiation of Th1 and Th2 cells in vitro depended on adenosine A2A receptors, which were also required for the efficacy of inosine and of L. reuteri in vivo. These results reveal that the microbiota–inosine–A2A receptor axis might represent a potential avenue for combatting autoimmune diseases mediated by T reg cell dysfunction. PMID:27994068

21 CFR 176.300 - Slimicides.

Code of Federal Regulations, 2013 CFR

2013-04-01

...) cyclohexanone 1,2-Bis(monobromoacetoxy) ethane [CA Reg. No. 3785-34-0] At a maximum level of 0.10 pound per ton... Methylenebisbutanethiolsulfonate Methylenebisthiocyanate 2-Nitrobutyl bromoacetate [CA Reg. No. 32815-96-6] At a maximum level of 0...)phosphonium sulfate (CAS Reg. No. 55566-30-8) Maximum use level of 84 mg/kg in the pulp slurry. The additive...

21 CFR 176.300 - Slimicides.

Code of Federal Regulations, 2012 CFR

2012-04-01

...) cyclohexanone 1,2-Bis(monobromoacetoxy) ethane [CA Reg. No. 3785-34-0] At a maximum level of 0.10 pound per ton... Methylenebisbutanethiolsulfonate Methylenebisthiocyanate 2-Nitrobutyl bromoacetate [CA Reg. No. 32815-96-6] At a maximum level of 0...)phosphonium sulfate (CAS Reg. No. 55566-30-8) Maximum use level of 84 mg/kg in the pulp slurry. The additive...

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing

DOEpatents

Church, George M.; Esvelt, Kevin; Mali, Prashant

2017-03-07

Methods of modulating expression of a target nucleic acid in a cell are provided including use of multiple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding genes or simultaneously and independently edit corresponding genes.

Therapeutic genome engineering via CRISPR-Cas systems.

PubMed

Moreno, Ana M; Mali, Prashant

2017-07-01

Differences in genomes underlie most organismal diversity, and aberrations in genomes underlie many disease states. With the growing knowledge of the genetic and pathogenic basis of human disease, development of safe and efficient platforms for genome and epigenome engineering will transform our ability to therapeutically target human diseases and also potentially engineer disease resistance. In this regard, the recent advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) RNA-guided nuclease systems have transformed our ability to target nucleic acids. Here we review therapeutic genome engineering applications with a specific focus on the CRISPR-Cas toolsets. We summarize past and current work, and also outline key challenges and future directions. WIREs Syst Biol Med 2017, 9:e1380. doi: 10.1002/wsbm.1380 For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.

RegA, an AraC-like protein, is a global transcriptional regulator that controls virulence gene expression in Citrobacter rodentium.

PubMed

Hart, Emily; Yang, Ji; Tauschek, Marija; Kelly, Michelle; Wakefield, Matthew J; Frankel, Gad; Hartland, Elizabeth L; Robins-Browne, Roy M

2008-11-01

Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5alpha, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization.

RegA, an AraC-Like Protein, Is a Global Transcriptional Regulator That Controls Virulence Gene Expression in Citrobacter rodentium▿

PubMed Central

Hart, Emily; Yang, Ji; Tauschek, Marija; Kelly, Michelle; Wakefield, Matthew J.; Frankel, Gad; Hartland, Elizabeth L.; Robins-Browne, Roy M.

2008-01-01

Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5α, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization. PMID:18765720

Targeted Delivery of CRISPR/Cas9-Mediated Cancer Gene Therapy via Liposome-Templated Hydrogel Nanoparticles.

PubMed

Chen, Zeming; Liu, Fuyao; Chen, Yanke; Liu, Jun; Wang, Xiaoying; Chen, Ann T; Deng, Gang; Zhang, Hongyi; Liu, Jie; Hong, Zhangyong; Zhou, Jiangbing

2017-12-08

Due to its simplicity, versatility, and high efficiency, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has emerged as one of the most promising approaches for treatment of a variety of genetic diseases, including human cancers. However, further translation of CRISPR/Cas9 for cancer gene therapy requires development of safe approaches for efficient, highly specific delivery of both Cas9 and single guide RNA to tumors. Here, novel core-shell nanostructure, liposome-templated hydrogel nanoparticles (LHNPs) that are optimized for efficient codelivery of Cas9 protein and nucleic acids is reported. It is demonstrated that, when coupled with the minicircle DNA technology, LHNPs deliver CRISPR/Cas9 with efficiency greater than commercial agent Lipofectamine 2000 in cell culture and can be engineered for targeted inhibition of genes in tumors, including tumors the brain. When CRISPR/Cas9 targeting a model therapeutic gene, polo-like kinase 1 (PLK1), is delivered, LHNPs effectively inhibit tumor growth and improve tumor-bearing mouse survival. The results suggest LHNPs as versatile CRISPR/Cas9-delivery tool that can be adapted for experimentally studying the biology of cancer as well as for clinically translating cancer gene therapy.

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.

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer.

PubMed

Richter, Corinna; Dy, Ron L; McKenzie, Rebecca E; Watson, Bridget N J; Taylor, Corinda; Chang, James T; McNeil, Matthew B; Staals, Raymond H J; Fineran, Peter C

2014-07-01

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼ 350 new spacers acquired in priming events and identified a 5'-protospacer-GG-3' protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2-3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Description and evaluation of the Earth System Regional Climate Model (RegCM-ES)

NASA Astrophysics Data System (ADS)

Farneti, Riccardo; Sitz, Lina; Di Sante, Fabio; Fuentes-Franco, Ramon; Coppola, Erika; Mariotti, Laura; Reale, Marco; Sannino, Gianmaria; Barreiro, Marcelo; Nogherotto, Rita; Giuliani, Graziano; Graffino, Giorgio; Solidoro, Cosimo; Giorgi, Filippo

2017-04-01

The increasing availability of satellite remote sensing data, of high temporal frequency and spatial resolution, has provided a new and enhanced view of the global ocean and atmosphere, revealing strong air-sea coupling processes throughout the ocean basins. In order to obtain an accurate representation and better understanding of the climate system, its variability and change, the inclusion of all mechanisms of interaction among the different sub-components, at high temporal and spatial resolution, becomes ever more desirable. Recently, global coupled models have been able to progressively refine their horizontal resolution to attempt to resolve smaller-scale processes. However, regional coupled ocean-atmosphere models can achieve even finer resolutions and provide additional information on the mechanisms of air-sea interactions and feedbacks. Here we describe a new, state-of-the-art, Earth System Regional Climate Model (RegCM-ES). RegCM-ES presently includes the coupling between atmosphere, ocean, land surface and sea-ice components, as well as an hydrological and ocean biogeochemistry model. The regional coupled model has been implemented and tested over some of the COordinated Regional climate Downscaling Experiment (CORDEX) domains. RegCM-ES has shown improvements in the representation of precipitation and SST fields over the tested domains, as well as realistic representations of coupled air-sea processes and interactions. The RegCM-ES model, which can be easily implemented over any regional domain of interest, is open source making it suitable for usage by the large scientific community.

CoryneRegNet: an ontology-based data warehouse of corynebacterial transcription factors and regulatory networks.

PubMed

Baumbach, Jan; Brinkrolf, Karina; Czaja, Lisa F; Rahmann, Sven; Tauch, Andreas

2006-02-14

The application of DNA microarray technology in post-genomic analysis of bacterial genome sequences has allowed the generation of huge amounts of data related to regulatory networks. This data along with literature-derived knowledge on regulation of gene expression has opened the way for genome-wide reconstruction of transcriptional regulatory networks. These large-scale reconstructions can be converted into in silico models of bacterial cells that allow a systematic analysis of network behavior in response to changing environmental conditions. CoryneRegNet was designed to facilitate the genome-wide reconstruction of transcriptional regulatory networks of corynebacteria relevant in biotechnology and human medicine. During the import and integration process of data derived from experimental studies or literature knowledge CoryneRegNet generates links to genome annotations, to identified transcription factors and to the corresponding cis-regulatory elements. CoryneRegNet is based on a multi-layered, hierarchical and modular concept of transcriptional regulation and was implemented by using the relational database management system MySQL and an ontology-based data structure. Reconstructed regulatory networks can be visualized by using the yFiles JAVA graph library. As an application example of CoryneRegNet, we have reconstructed the global transcriptional regulation of a cellular module involved in SOS and stress response of corynebacteria. CoryneRegNet is an ontology-based data warehouse that allows a pertinent data management of regulatory interactions along with the genome-scale reconstruction of transcriptional regulatory networks. These models can further be combined with metabolic networks to build integrated models of cellular function including both metabolism and its transcriptional regulation.

Bicarbonate-mediated transcriptional activation of divergent operons by the virulence regulatory protein, RegA, from Citrobacter rodentium.

PubMed

Yang, Ji; Hart, Emily; Tauschek, Marija; Price, G Dean; Hartland, Elizabeth L; Strugnell, Richard A; Robins-Browne, Roy M

2008-04-01

Regulation of virulence gene expression plays a central role in the pathogenesis of enteric bacteria as they encounter diverse environmental conditions in the gastrointestinal tract of their hosts. In this study, we investigated environmental regulation of two putative virulence determinants adcA and kfc by RegA, an AraC/XylS-like regulator, from Citrobacter rodentium, and identified bicarbonate as the environmental signal which induced transcription of adcA and kfc through RegA. Primer extension experiments showed that adcA and kfc were divergently transcribed from sigma(70) promoters. In vivo and in vitro experiments demonstrated that bicarbonate facilitated and stabilized the binding of RegA to an operator located between the two promoters. The interaction of RegA with its DNA target resulted in the formation of a nucleosome-like structure, which evidently displaced the histone-like proteins, H-NS and StpA, from the adcA and kfc promoter regions, leading to transcriptional derepression. In addition, our results indicated that RegA also behaved as a Class I activator by directly stimulating transcription initiation by RNA polymerase. This is the first report to describe the molecular mechanism by which an environmental chemical stimulates transcription of virulence-associated genes of an enteric pathogen through an AraC/XlyS-like activator.

The role of CRISPR-Cas systems in virulence of pathogenic bacteria.

PubMed

Louwen, Rogier; Staals, Raymond H J; Endtz, Hubert P; van Baarlen, Peter; van der Oost, John

2014-03-01

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been used as a complementary typing tool in diagnostic, epidemiologic, and evolutionary analyses of prokaryotic strains. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. Key elements of this unique prokaryotic defense system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading viruses and plasmids, generally followed by the degradation of the invader. In addition, several recent studies have pointed at direct links of CRISPR-Cas to regulation of a range of stress-related phenomena. An interesting example concerns a pathogenic bacterium that possesses a CRISPR-associated ribonucleoprotein complex that may play a dual role in defense and/or virulence. In this review, we describe recently reported cases of potential involvement of CRISPR-Cas systems in bacterial stress responses in general and bacterial virulence in particular.

Intestinal REG3 Lectins Protect Against Alcoholic Steatohepatitis by Reducing Mucosa-Associated Microbiota and Preventing Bacterial Translocation

PubMed Central

Wang, Lirui; Fouts, Derrick E.; Stärkel, Peter; Hartmann, Phillipp; Chen, Peng; Llorente, Cristina; DePew, Jessica; Moncera, Kelvin; Ho, Samuel B.; Brenner, David A.; Hooper, Lora V.; Schnabl, Bernd

2016-01-01

Summary Approximately half of all deaths from liver cirrhosis, the 10th leading cause of mortality in the United States, are related to alcohol use. Chronic alcohol consumption is accompanied by intestinal dysbiosis and bacterial overgrowth, yet little is known about the factors that alter the microbial composition or their contribution to liver disease. We previously associated chronic alcohol consumption with lower intestinal levels of the antimicrobial-regenerating islet-derived (REG)-3 lectins. Here, we demonstrate that intestinal deficiency in REG3B or REG3G increases numbers of mucosa-associated bacteria and enhances bacterial translocation to the mesenteric lymph nodes and liver, promoting the progression of ethanol-induced fatty liver disease toward steatohepatitis. Overexpression of Reg3g in intestinal epithelial cells restricts bacterial colonization of mucosal surfaces, reduces bacterial translocation, and protects mice from alcohol-induced steatohepatitis. Thus, alcohol appears to impair control of the mucosa-associated microbiota, and subsequent breach of the mucosal barrier facilitates progression of alcoholic liver disease. PMID:26867181

RegCM4-HadGEM2-ES simulated cyclone climatology (1979-2005) over the Southwestern South Atlantic Ocean

NASA Astrophysics Data System (ADS)

Porfírio da Rocha, Rosmeri; Simões Reboita, Michelle

2015-04-01

Cyclones over the Southwestern South Atlantic Ocean (SAO) are a subject of great interest once they modify the weather and control the climate near east coast of South America (SA). In this study we compare the cyclones climatology in the period 1979-2005 simulated by Regional Climate Model version 4 (RegCM4) with that from ERA-Interim reanalysis (ECMWF). RegCM4 was nested in HadGEM2-ES output and the simulation used the SA domain of CORDEX project, with a horizontal grid of 50 km and 18 sigma-pressure levels in the vertical. The RegCM4 simulation used the land surface Biosphere-Atmosphere Transfer Scheme (BATS) and the mixed convection Emanuel-Grell scheme configurations. This simulation is part of the CREMA (CORDEX REgCM4 hyper-MAtrix) experiment. The cyclones were identified using an automated tracking scheme based on minima (cyclonic in Southern Hemisphere) of relative vorticity from the wind at 925 hPa. The threshold of -1.5 x 10-5s-1 was used in the algorithm. All cyclones in RegCM4 and ERA-Interim with relative vorticity lower than this threshold and with lifetime higher or equal 24 hours were included in the climatology. ERA-Interim shows three main cyclogenetic regions near east coast of SA. In general, RegCM4 simulated these same regions but with an underestimation of the number of cyclones. In each of these regions, there is a different season of higher cyclones frequency. Over extreme south of southern Brazil and Uruguay the higher frequency of cyclones occurs in winter, while southeastern Brazil and southeastern Argentina cyclones are most frequent during summer. RegCM4 is able to simulate this observed seasonality.

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...

Assessment of cerebral blood flow autoregulation (CBF AR) with rheoencephalography (REG): studies in animals

NASA Astrophysics Data System (ADS)

Popovic, Djordje; Bodo, Michael; Pearce, Frederick; van Albert, Stephen; Garcia, Alison; Settle, Tim; Armonda, Rocco

2013-04-01

The ability of cerebral vasculature to regulate cerebral blood flow (CBF) in the face of changes in arterial blood pressure (SAP) or intracranial pressure (ICP) is an important guard against secondary ischemia in acute brain injuries, and official guidelines recommend that therapeutic decisions be guided by continuous monitoring of CBF autoregulation (AR). The common method for CBF AR monitoring, which rests on real-time derivation of the correlation coefficient (PRx) between slow oscillations in SAP and ICP is, however, rarely used in clinical practice because it requires invasive ICP measurements. This study investigated whether the correlation coefficient between SAP and the pulsatile component of the non-invasive transcranial bioimpedance signal (rheoencephalography, REG) could be used to assess the state and lower limit of CBF AR. The results from pigs and rhesus macaques affirm the utility of REG; however, additional animal and clinical studies are warranted to assess selectivity of automatic REG-based evaluation of CBF AR.

CoryneRegNet: An ontology-based data warehouse of corynebacterial transcription factors and regulatory networks

PubMed Central

Baumbach, Jan; Brinkrolf, Karina; Czaja, Lisa F; Rahmann, Sven; Tauch, Andreas

2006-01-01

Background The application of DNA microarray technology in post-genomic analysis of bacterial genome sequences has allowed the generation of huge amounts of data related to regulatory networks. This data along with literature-derived knowledge on regulation of gene expression has opened the way for genome-wide reconstruction of transcriptional regulatory networks. These large-scale reconstructions can be converted into in silico models of bacterial cells that allow a systematic analysis of network behavior in response to changing environmental conditions. Description CoryneRegNet was designed to facilitate the genome-wide reconstruction of transcriptional regulatory networks of corynebacteria relevant in biotechnology and human medicine. During the import and integration process of data derived from experimental studies or literature knowledge CoryneRegNet generates links to genome annotations, to identified transcription factors and to the corresponding cis-regulatory elements. CoryneRegNet is based on a multi-layered, hierarchical and modular concept of transcriptional regulation and was implemented by using the relational database management system MySQL and an ontology-based data structure. Reconstructed regulatory networks can be visualized by using the yFiles JAVA graph library. As an application example of CoryneRegNet, we have reconstructed the global transcriptional regulation of a cellular module involved in SOS and stress response of corynebacteria. Conclusion CoryneRegNet is an ontology-based data warehouse that allows a pertinent data management of regulatory interactions along with the genome-scale reconstruction of transcriptional regulatory networks. These models can further be combined with metabolic networks to build integrated models of cellular function including both metabolism and its transcriptional regulation. PMID:16478536

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.

CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum.

PubMed

Cho, Jae Sung; Choi, Kyeong Rok; Prabowo, Cindy Pricilia Surya; Shin, Jae Ho; Yang, Dongsoo; Jang, Jaedong; Lee, Sang Yup

2017-07-01

Genome engineering of Corynebacterium glutamicum, an important industrial microorganism for amino acids production, currently relies on random mutagenesis and inefficient double crossover events. Here we report a rapid genome engineering strategy to scarlessly knock out one or more genes in C. glutamicum in sequential and iterative manner. Recombinase RecT is used to incorporate synthetic single-stranded oligodeoxyribonucleotides into the genome and CRISPR/Cas9 to counter-select negative mutants. We completed the system by engineering the respective plasmids harboring CRISPR/Cas9 and RecT for efficient curing such that multiple gene targets can be done iteratively and final strains will be free of plasmids. To demonstrate the system, seven different mutants were constructed within two weeks to study the combinatorial deletion effects of three different genes on the production of γ-aminobutyric acid, an industrially relevant chemical of much interest. This genome engineering strategy will expedite metabolic engineering of C. glutamicum. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Investigating CRISPR-Cas systems in Clostridium botulinum via bioinformatics tools.

PubMed

Negahdaripour, Manica; Nezafat, Navid; Hajighahramani, Nasim; Rahmatabadi, Seyyed Soheil; Ghasemi, Younes

2017-10-01

The Clustered regularly interspaced short palindromic repeats (CRISPR) systems are a type of innate immunity found in some prokaryotes, which protect them against alien genetic elements by targeting foreign nucleic acids. Some other functions are also attributed to these systems. Clostridium botulinum bacteria produce botulinum neurotoxins (BoNT), one of the deadliest known toxins for humans and some animals. Food poisoning due to these bacteria is still a challenge in food industries. On the other hand, BoNT has been widely investigated for therapeutic applications including different muscle disorders. Bont genes may be located on bacterial chromosomes, plasmids, or even prophages. Generally, the genomes of Cl. botulinum show a high level of plasticity. In order to investigate the presence and characteristics of CRISPRs in these anaerobe bacteria, an in silico study on 113 CRISPR arrays identified in 38 Cl. botulinum strains was performed. A high occurrence of CRISPR arrays (80%) were found, with a remarkable frequency on plasmids. Several (CRISPR-associated) Cas proteins from different types were recognized in the studied strains, which were mostly Cas6. The CRISPR-Cas systems were identified as type I or III, but no type II. The spacers showed more homology with bacterial plasmids than phages. Active CRISPR-Cas systems can prevent the transfer of foreign genes, which may also include bont genes. This study provides the first insight into the probable roles of CRISPR-Cas systems in Cl. botulinum strains such as toxigenicity. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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.

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.

CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.

PubMed

Deng, Wulan; Shi, Xinghua; Tjian, Robert; Lionnet, Timothée; Singer, Robert H

2015-09-22

Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

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

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.

CoryneRegNet 4.0 – A reference database for corynebacterial gene regulatory networks

PubMed Central

Baumbach, Jan

2007-01-01

Background Detailed information on DNA-binding transcription factors (the key players in the regulation of gene expression) and on transcriptional regulatory interactions of microorganisms deduced from literature-derived knowledge, computer predictions and global DNA microarray hybridization experiments, has opened the way for the genome-wide analysis of transcriptional regulatory networks. The large-scale reconstruction of these networks allows the in silico analysis of cell behavior in response to changing environmental conditions. We previously published CoryneRegNet, an ontology-based data warehouse of corynebacterial transcription factors and regulatory networks. Initially, it was designed to provide methods for the analysis and visualization of the gene regulatory network of Corynebacterium glutamicum. Results Now we introduce CoryneRegNet release 4.0, which integrates data on the gene regulatory networks of 4 corynebacteria, 2 mycobacteria and the model organism Escherichia coli K12. As the previous versions, CoryneRegNet provides a web-based user interface to access the database content, to allow various queries, and to support the reconstruction, analysis and visualization of regulatory networks at different hierarchical levels. In this article, we present the further improved database content of CoryneRegNet along with novel analysis features. The network visualization feature GraphVis now allows the inter-species comparisons of reconstructed gene regulatory networks and the projection of gene expression levels onto that networks. Therefore, we added stimulon data directly into the database, but also provide Web Service access to the DNA microarray analysis platform EMMA. Additionally, CoryneRegNet now provides a SOAP based Web Service server, which can easily be consumed by other bioinformatics software systems. Stimulons (imported from the database, or uploaded by the user) can be analyzed in the context of known transcriptional regulatory networks to

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

RegNetwork: an integrated database of transcriptional and post-transcriptional regulatory networks in human and mouse

PubMed Central

Liu, Zhi-Ping; Wu, Canglin; Miao, Hongyu; Wu, Hulin

2015-01-01

Transcriptional and post-transcriptional regulation of gene expression is of fundamental importance to numerous biological processes. Nowadays, an increasing amount of gene regulatory relationships have been documented in various databases and literature. However, to more efficiently exploit such knowledge for biomedical research and applications, it is necessary to construct a genome-wide regulatory network database to integrate the information on gene regulatory relationships that are widely scattered in many different places. Therefore, in this work, we build a knowledge-based database, named ‘RegNetwork’, of gene regulatory networks for human and mouse by collecting and integrating the documented regulatory interactions among transcription factors (TFs), microRNAs (miRNAs) and target genes from 25 selected databases. Moreover, we also inferred and incorporated potential regulatory relationships based on transcription factor binding site (TFBS) motifs into RegNetwork. As a result, RegNetwork contains a comprehensive set of experimentally observed or predicted transcriptional and post-transcriptional regulatory relationships, and the database framework is flexibly designed for potential extensions to include gene regulatory networks for other organisms in the future. Based on RegNetwork, we characterized the statistical and topological properties of genome-wide regulatory networks for human and mouse, we also extracted and interpreted simple yet important network motifs that involve the interplays between TF-miRNA and their targets. In summary, RegNetwork provides an integrated resource on the prior information for gene regulatory relationships, and it enables us to further investigate context-specific transcriptional and post-transcriptional regulatory interactions based on domain-specific experimental data. Database URL: http://www.regnetworkweb.org PMID:26424082

RegPrecise 3.0--a resource for genome-scale exploration of transcriptional regulation in bacteria.

PubMed

Novichkov, Pavel S; Kazakov, Alexey E; Ravcheev, Dmitry A; Leyn, Semen A; Kovaleva, Galina Y; Sutormin, Roman A; Kazanov, Marat D; Riehl, William; Arkin, Adam P; Dubchak, Inna; Rodionov, Dmitry A

2013-11-01

Genome-scale prediction of gene regulation and reconstruction of transcriptional regulatory networks in prokaryotes is one of the critical tasks of modern genomics. Bacteria from different taxonomic groups, whose lifestyles and natural environments are substantially different, possess highly diverged transcriptional regulatory networks. The comparative genomics approaches are useful for in silico reconstruction of bacterial regulons and networks operated by both transcription factors (TFs) and RNA regulatory elements (riboswitches). RegPrecise (http://regprecise.lbl.gov) is a web resource for collection, visualization and analysis of transcriptional regulons reconstructed by comparative genomics. We significantly expanded a reference collection of manually curated regulons we introduced earlier. RegPrecise 3.0 provides access to inferred regulatory interactions organized by phylogenetic, structural and functional properties. Taxonomy-specific collections include 781 TF regulogs inferred in more than 160 genomes representing 14 taxonomic groups of Bacteria. TF-specific collections include regulogs for a selected subset of 40 TFs reconstructed across more than 30 taxonomic lineages. Novel collections of regulons operated by RNA regulatory elements (riboswitches) include near 400 regulogs inferred in 24 bacterial lineages. RegPrecise 3.0 provides four classifications of the reference regulons implemented as controlled vocabularies: 55 TF protein families; 43 RNA motif families; ~150 biological processes or metabolic pathways; and ~200 effectors or environmental signals. Genome-wide visualization of regulatory networks and metabolic pathways covered by the reference regulons are available for all studied genomes. A separate section of RegPrecise 3.0 contains draft regulatory networks in 640 genomes obtained by an conservative propagation of the reference regulons to closely related genomes. RegPrecise 3.0 gives access to the transcriptional regulons reconstructed in

Chromosomal targeting by CRISPR-Cas systems can contribute to genome plasticity in bacteria

PubMed Central

Dy, Ron L; Pitman, Andrew R; Fineran, Peter C

2013-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR) and their associated (Cas) proteins form adaptive immune systems in bacteria to combat phage and other foreign genetic elements. Typically, short spacer sequences are acquired from the invader DNA and incorporated into CRISPR arrays in the bacterial genome. Small RNAs are generated that contain these spacer sequences and enable sequence-specific destruction of the foreign nucleic acids. Occasionally, spacers are acquired from the chromosome, which instead leads to targeting of the host genome. Chromosomal targeting is highly toxic to the bacterium, providing a strong selective pressure for a variety of evolutionary routes that enable host cell survival. Mutations that inactivate the CRISPR-Cas functionality, such as within the cas genes, CRISPR repeat, protospacer adjacent motifs (PAM), and target sequence, mediate escape from toxicity. This self-targeting might provide some explanation for the incomplete distribution of CRISPR-Cas systems in less than half of sequenced bacterial genomes. More importantly, self-genome targeting can cause large-scale genomic alterations, including remodeling or deletion of pathogenicity islands and other non-mobile chromosomal regions. While control of horizontal gene transfer is perceived as their main function, our recent work illuminates an alternative role of CRISPR-Cas systems in causing host genomic changes and influencing bacterial evolution. PMID:24251073

Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.

PubMed

Gao, Hanchao; Zhao, Chengjiang; Xiang, Xi; Li, Yong; Zhao, Yanli; Li, Zesong; Pan, Dengke; Dai, Yifan; Hara, Hidetaka; Cooper, David K C; Cai, Zhiming; Mou, Lisha

2017-02-16

Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

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.

The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

PubMed Central

Staals, Raymond H. J.; Endtz, Hubert P.; van Baarlen, Peter; van der Oost, John

2014-01-01

SUMMARY Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been used as a complementary typing tool in diagnostic, epidemiologic, and evolutionary analyses of prokaryotic strains. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. Key elements of this unique prokaryotic defense system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading viruses and plasmids, generally followed by the degradation of the invader. In addition, several recent studies have pointed at direct links of CRISPR-Cas to regulation of a range of stress-related phenomena. An interesting example concerns a pathogenic bacterium that possesses a CRISPR-associated ribonucleoprotein complex that may play a dual role in defense and/or virulence. In this review, we describe recently reported cases of potential involvement of CRISPR-Cas systems in bacterial stress responses in general and bacterial virulence in particular. PMID:24600041

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.

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.

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.

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein.

PubMed

Pawluk, April; Shah, Megha; Mejdani, Marios; Calmettes, Charles; Moraes, Trevor F; Davidson, Alan R; Maxwell, Karen L

2017-12-12

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system. Copyright © 2017 Pawluk et al.

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.

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

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

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

PubMed Central

Shah, Megha; Mejdani, Marios; Calmettes, Charles; Moraes, Trevor F.

2017-01-01

ABSTRACT CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor. PMID:29233895

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.

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

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

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.

Study on analysis of waste edible oil with deterioration and removal of acid value, carbonyl value, and free fatty acid by a food additive (calcium silicate).

PubMed

Ogata, Fumihiko; Tanaka, Yuko; Tominaga, Hisato; Kangawa, Moe; Inoue, Kenji; Ueda, Ayaka; Iwata, Yuka; Kawasaki, Naohito

2013-01-01

This study investigated the regeneration of waste edible oil using a food additive (calcium silicate, CAS). Waste edible oil was prepared by combined heat and aeration treatment. Moreover, the deterioration of edible oil by combined heat and aeration treatment was greater than that by heat treatment alone. The acid value (AV) and carbonyl value (CV) increased with increasing deterioration; conversely, the tocopherol concentration decreased with increasing deterioration. The specific surface area, pore volume, and mean pore diameter of the 3 CAS formulations used (CAS30, CAS60, and CAS90) were evaluated, and scanning electron microscopic images were taken. The specific surface area increased in the order of CAS30 (115.54 m(2)/g) < CAS60 (163.93 m(2)/g) < CAS90 (187.47 m(2)/g). The mean pore diameter increased in the order of CAS90 (170.59 Å) < CAS60 (211.60 Å) < CAS30 (249.70 Å). The regeneration of waste edible oil was possible with CAS treatment. The AV reduced by 15.2%, 10.8%, and 23.1% by CAS30, CAS60, and CAS90 treatment, respectively, and the CV was reduced by 35.6%, 29.8%, and 31.3% by these 3 treatments, respectively. Moreover, the concentrations of tocopherol and free fatty acids did not change with CAS treatment. The characteristics of CAS were not related to the degree of change of AV and CV. However, the adsorption mechanism of polar and non-polar compounds generated in waste edible oil by CAS was related with the presence of silica gel molecules in CAS. The findings indicated that CAS was useful for the regeneration of waste edible oil.

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.

Interaction of cinnamic acid derivatives with β-cyclodextrin in water: experimental and molecular modeling studies.

PubMed

Liu, Benguo; Zeng, Jie; Chen, Chen; Liu, Yonglan; Ma, Hanjun; Mo, Haizhen; Liang, Guizhao

2016-03-01

Cyclodextrins (CDs) can be used to improve the solubility and stability of cinnamic acid derivatives (CAs). However, there was no detailed report about understanding the effects of the substituent groups in the benzene ring on the inclusion behavior between CAs and CDs in aqueous solution. Here, the interaction of β-CD with CAs, including caffeic acid, ferulic acid, and p-coumaric acid, in water was investigated by phase-solubility method, UV, fluorescence, and (1)H NMR spectroscopy, together with ONIOM (our Own N-layer Integrated Orbital molecular Mechanics)-based QM/MM (Quantum Mechanics/Molecular Mechanics) calculations. Experimental results demonstrated that CAs could form 1:1 stoichiometric inclusion complex with β-CD by non-covalent bonds, and that the maximum apparent stability constants were found in caffeic acid (176M(-1)) followed by p-coumaric acid (160M(-1)) and ferulic acid (133M(-1)). Moreover, our calculations reasonably illustrated the binding orientations of β-CD with CAs determined by experimental observations. Copyright © 2015. Published by Elsevier Ltd.

A complex of Cas proteins 5, 6, and 7 is required for the biogenesis and stability of clustered regularly interspaced short palindromic repeats (crispr)-derived rnas (crrnas) in Haloferax volcanii.

PubMed

Brendel, Jutta; Stoll, Britta; Lange, Sita J; Sharma, Kundan; Lenz, Christof; Stachler, Aris-Edda; Maier, Lisa-Katharina; Richter, Hagen; Nickel, Lisa; Schmitz, Ruth A; Randau, Lennart; Allers, Thorsten; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita

2014-03-07

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1-8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.

The regional climate model RegCM3 performances over several regions and climate regimes

NASA Astrophysics Data System (ADS)

Coppola, E.; Rauscher, S.; Gao, X.; Giorgi, F.; Im, E. S.; Mariotti, L.; Seth, A.; Sylla, M. B.

2009-04-01

Regional Climate models are more and more needed to provide high resolution regional climate information in climate impact studies. Water availability in a future scenario is the main request of policy makers for adaptation and mitigation purposes. However precipitation changes are unlikely to be as spatially coherent as temperature changes and they are closely related to the regional model itself. In addition model skill varies regionally. An example of several ICTP regional climate model (RegCM3) simulations is reported over China, Korea, Africa, Central and Southern America, Europe and Australia. Over China, Australia, and Korea the regional model improves the simulation compared to the driving GCM when compared with CRU observations. In China, for example, the higher resolution of the regional model inhibits the penetration of the monsoon precipitation front from the southern slope of the Himalaya onto the Tibetan Plateau. In Korea the nested domain simulation (20 km) shows an encouraging performance with regard to capturing extreme precipitation episodes and the finer spatial distribution reflects the detailed geography of the Korean Peninsula. Over South America, RegCM captures the annual cycle of precipitation over Northeast Brazil and the South American Monsoon region, although the monsoon onset occurs too early in the model. Precipitation over the Amazon is not well captured, with too little precipitation associated with weak easterlies and reduced moisture transport into the interior of the continent. RegCM simulates the annual cycle of precipitation over Central America and the Caribbean fairly well; in particular, the complex spatial distribution of the Mid-Summer Drought, a decrease in precipitation that occurs during the middle of the rainy season in July and August, is better captured by RegCM than by the GCM. In addition, RegCM simulates the strength and position of the Caribbean low level jet, a mesoscale feature related to precipitation anomalies

75 FR 5850 - Proposed Collection; Comment Request for REG-166012-02

Federal Register 2010, 2011, 2012, 2013, 2014

2010-02-04

... 1995, Public Law 104-13 (44 U.S.C. 3506(c)(2)(A)). Currently, the IRS is soliciting comments concerning REG-166012-02, Notional Principal Contracts; Contingent Nonperiodic Payments. DATES: Written [email protected] . SUPPLEMENTARY INFORMATION: Title: Notional Principal Contracts; Contingent Nonperiodic...

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.

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…

Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome.

PubMed

Zhen, Shuai; Takahashi, Yoichiro; Narita, Shunichi; Yang, Yi-Chen; Li, Xu

2017-02-07

The potent ability of CRISPR/Cas9 system to inhibit the expression of targeted gene is being exploited as a new class of therapeutics for a variety of diseases. However, the efficient and safe delivery of CRISPR/Cas9 into specific cell populations is still the principal challenge in the clinical development of CRISPR/Cas9 therapeutics. In this study, a flexible aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery and increased flexibility. Our chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand. Cationic liposomes were linked to aptamers by the post-insertion method and were used to deliver therapeutic CRISPR/Cas9 that target the survival gene, polo-like kinase 1, in tumor cells. We demonstrate that the aptamer-liposome-CRISPR/Cas9 chimeras had a significant cell-type binding specificity and a remarkable gene silencing effect in vitro. Furthermore, silencing promoted a conspicuous regression of prostate cancer in vivo. Importantly, the approach described here provides a universal means of cell type-specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs.

Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome

PubMed Central

Zhen, Shuai; Takahashi, Yoichiro; Narita, Shunichi; Yang, Yi-Chen; Li, Xu

2017-01-01

The potent ability of CRISPR/Cas9 system to inhibit the expression of targeted gene is being exploited as a new class of therapeutics for a variety of diseases. However, the efficient and safe delivery of CRISPR/Cas9 into specific cell populations is still the principal challenge in the clinical development of CRISPR/Cas9 therapeutics. In this study, a flexible aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery and increased flexibility. Our chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand. Cationic liposomes were linked to aptamers by the post-insertion method and were used to deliver therapeutic CRISPR/Cas9 that target the survival gene, polo-like kinase 1, in tumor cells. We demonstrate that the aptamer-liposome-CRISPR/Cas9 chimeras had a significant cell-type binding specificity and a remarkable gene silencing effect in vitro. Furthermore, silencing promoted a conspicuous regression of prostate cancer in vivo. Importantly, the approach described here provides a universal means of cell type–specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs. PMID:28030843

[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…

A Complex of Cas Proteins 5, 6, and 7 Is Required for the Biogenesis and Stability of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii*

PubMed Central

Brendel, Jutta; Stoll, Britta; Lange, Sita J.; Sharma, Kundan; Lenz, Christof; Stachler, Aris-Edda; Maier, Lisa-Katharina; Richter, Hagen; Nickel, Lisa; Schmitz, Ruth A.; Randau, Lennart; Allers, Thorsten; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita

2014-01-01

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1–8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA. PMID:24459147

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.

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

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.

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.

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

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.

Mycobacterium tuberculosis PhoY Proteins Promote Persister Formation by Mediating Pst/SenX3-RegX3 Phosphate Sensing.

PubMed

Namugenyi, Sarah B; Aagesen, Alisha M; Elliott, Sarah R; Tischler, Anna D

2017-07-11

The Mycobacterium tuberculosis phosphate-specific transport (Pst) system controls gene expression in response to phosphate availability by inhibiting the activation of the SenX3-RegX3 two-component system under phosphate-rich conditions, but the mechanism of communication between these systems is unknown. In Escherichia coli , inhibition of the two-component system PhoR-PhoB under phosphate-rich conditions requires both the Pst system and PhoU, a putative adaptor protein. E. coli PhoU is also involved in the formation of persisters, a subpopulation of phenotypically antibiotic-tolerant bacteria. M. tuberculosis encodes two PhoU orthologs, PhoY1 and PhoY2. We generated phoY single- and double-deletion mutants and examined the expression of RegX3-regulated genes by quantitative reverse transcription-PCR (qRT-PCR). Gene expression was increased only in the Δ phoY1 Δ phoY2 double mutant and could be restored to the wild-type level by complementation with either phoY1 or phoY2 or by deletion of regX3 These data suggest that the PhoY proteins function redundantly to inhibit SenX3-RegX3 activation. We analyzed the frequencies of antibiotic-tolerant persister variants in the phoY mutants using several antibiotic combinations. Persister frequency was decreased at least 40-fold in the Δ phoY1 Δ phoY2 mutant compared to the frequency in the wild type, and this phenotype was RegX3 dependent. A Δ pstA1 mutant lacking a Pst system transmembrane component exhibited a similar RegX3-dependent decrease in persister frequency. In aerosol-infected mice, the Δ phoY1 Δ phoY2 and Δ pstA1 mutants were more susceptible to treatment with rifampin but not isoniazid. Our data demonstrate that disrupting phosphate sensing mediated by the PhoY proteins and the Pst system enhances the susceptibility of M. tuberculosis to antibiotics both in vitro and during infection. IMPORTANCE Persister variants, subpopulations of bacteria that are phenotypically antibiotic tolerant, contribute to

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.

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...

Rosebud SynCoal Partnership, SynCoal{reg_sign} demonstration technology update

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

Sheldon, R.W.

1997-12-31

An Advanced Coal Conversion Process (ACCP) technology being demonstrated in eastern Montana (USA) at the heart of one of the world`s largest coal deposits is providing evidence that the molecular structure of low-rank coals can be altered successfully to produce a unique product for a variety of utility and industrial applications. The product is called SynCoal{reg_sign} and the process has been developed by the Rosebud SynCoal Partnership (RSCP) through the US Department of Energy`s multi-million dollar Clean Coal Technology Program. The ACCP demonstration process uses low-pressure, superheated gases to process coal in vibrating fluidized beds. Two vibratory fluidized processing stagesmore » are used to heat and convert the coal. This is followed by a water spray quench and a vibratory fluidized stage to cool the coal. Pneumatic separators remove the solid impurities from the dried coal. There are three major steps to the SynCoal{reg_sign} process: (1) thermal treatment of the coal in an inert atmosphere, (2) inert gas cooling of the hot coal, and (3) removal of ash minerals. When operated continuously, the demonstration plant produces over 1,000 tons per day (up to 300,000 tons per year) of SynCoal{reg_sign} with a 2% moisture content, approximately 11,800b Btu/lb and less than 1.0 pound of SO{sub 2} per million Btu. This product is obtained from Rosebud Mine sub-bituminous coal which starts with 25% moisture, 8,600 Btu/lb and approximately 1.6 pounds of SO{sub 2} per million Btu.« less

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.

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.

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.

Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10.

PubMed

Bari, S M Nayeemul; Walker, Forrest C; Cater, Katie; Aslan, Barbaros; Hatoum-Aslan, Asma

2017-12-15

Staphylococci are prevalent skin-dwelling bacteria that are also leading causes of antibiotic-resistant infections. Viruses that infect and lyse these organisms (virulent staphylococcal phages) can be used as alternatives to conventional antibiotics and represent promising tools to eliminate or manipulate specific species in the microbiome. However, since over half their genes have unknown functions, virulent staphylococcal phages carry inherent risk to cause unknown downstream side effects. Further, their swift and destructive reproductive cycle make them intractable by current genetic engineering techniques. CRISPR-Cas10 is an elaborate prokaryotic immune system that employs small RNAs and a multisubunit protein complex to detect and destroy phages and other foreign nucleic acids. Some staphylococci naturally possess CRISPR-Cas10 systems, thus providing an attractive tool already installed in the host chromosome to harness for phage genome engineering. However, the efficiency of CRISPR-Cas10 immunity against virulent staphylococcal phages and corresponding utility as a tool to facilitate their genome editing has not been explored. Here, we show that the CRISPR-Cas10 system native to Staphylococcus epidermidis exhibits robust immunity against diverse virulent staphylococcal phages. On the basis of this activity, a general two-step approach was developed to edit these phages that relies upon homologous recombination machinery encoded in the host. Variations of this approach to edit toxic phage genes and access phages that infect CRISPR-less staphylococci are also presented. This versatile set of genetic tools enables the systematic study of phage genes of unknown functions and the design of genetically defined phage-based antimicrobials that can eliminate or manipulate specific Staphylococcus species.

On the ability of RegCM4 regional climate model to simulate surface solar radiation patterns over Europe: an assessment using satellite-based observations

NASA Astrophysics Data System (ADS)

Alexandri, G.; Georgoulias, A. K.; Zanis, P.; Katragkou, E.; Tsikerdekis, A.; Kourtidis, K.; Meleti, C.

2015-11-01

In this work, we assess the ability of RegCM4 regional climate model to simulate surface solar radiation (SSR) patterns over Europe. A decadal RegCM4 run (2000-2009) was implemented and evaluated against satellite-based observations from the Satellite Application Facility on Climate Monitoring (CM SAF), showing that the model simulates adequately the SSR patterns over the region. The SSR bias between RegCM4 and CM SAF is +1.5 % for MFG (Meteosat First Generation) and +3.3 % for MSG (Meteosat Second Generation) observations. The relative contribution of parameters that determine the transmission of solar radiation within the atmosphere to the deviation appearing between RegCM4 and CM SAF SSR is also examined. Cloud macrophysical and microphysical properties such as cloud fractional cover (CFC), cloud optical thickness (COT) and cloud effective radius (Re) from RegCM4 are evaluated against data from CM SAF. Generally, RegCM4 underestimates CFC by 24.3 % and Re for liquid/ice clouds by 36.1 %/28.3 % and overestimates COT by 4.3 %. The same procedure is repeated for aerosol optical properties such as aerosol optical depth (AOD), asymmetry factor (ASY) and single-scattering albedo (SSA), as well as other parameters, including surface broadband albedo (ALB) and water vapor amount (WV), using data from MACv1 aerosol climatology, from CERES satellite sensors and from ERA-Interim reanalysis. It is shown here that the good agreement between RegCM4 and satellite-based SSR observations can be partially attributed to counteracting effects among the above mentioned parameters. The potential contribution of each parameter to the RegCM4-CM SAF SSR deviations is estimated with the combined use of the aforementioned data and a~radiative transfer model (SBDART). CFC, COT and AOD are the major determinants of these deviations on a monthly basis; however, the other parameters also play an important role for specific regions and seasons. Overall, for the European domain, CFC, COT and

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

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.

21 CFR 173.342 - Chlorofluorocarbon 113 and perfluorohexane.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Chlorofluorocarbon 113 and perfluorohexane. 173... perfluorohexane. A mixture of 99 percent chlorofluorocarbon 113 (1,1,2-trichloro-1,2,2-trifluoroethane) (CAS Reg. No. 76-13-1, also known as fluorocarbon 113, CFC 113 and FC 113) and 1 percent perfluorohexane (CAS...

21 CFR 173.342 - Chlorofluorocarbon 113 and perfluorohexane.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Chlorofluorocarbon 113 and perfluorohexane. 173... perfluorohexane. A mixture of 99 percent chlorofluorocarbon 113 (1,1,2-trichloro-1,2,2-trifluoroethane) (CAS Reg. No. 76-13-1, also known as fluorocarbon 113, CFC 113 and FC 113) and 1 percent perfluorohexane (CAS...

21 CFR 173.342 - Chlorofluorocarbon 113 and perfluorohexane.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Chlorofluorocarbon 113 and perfluorohexane. 173... perfluorohexane. A mixture of 99 percent chlorofluorocarbon 113 (1,1,2-trichloro-1,2,2-trifluoroethane) (CAS Reg. No. 76-13-1, also known as fluorocarbon 113, CFC 113 and FC 113) and 1 percent perfluorohexane (CAS...

Intra-field on-product overlay improvement by application of RegC and TWINSCAN corrections

NASA Astrophysics Data System (ADS)

Sharoni, Ofir; Dmitriev, Vladimir; Graitzer, Erez; Perets, Yuval; Gorhad, Kujan; van Haren, Richard; Cekli, Hakki E.; Mulkens, Jan

2015-03-01

The on product overlay specification and Advanced Process Control (APC) is getting extremely challenging particularly after the introduction of multi-patterning applications like Spacer Assisted Double Patterning (SADP) and multipatterning techniques like N-repetitive Litho-Etch steps (LEN, N >= 2). When the latter is considered, most of the intrafield overlay contributors drop out of the overlay budget. This is a direct consequence of the fact that the scanner settings (like dose, illumination settings, etc.) as well as the subsequent processing steps can be made very similar for two consecutive Litho-Etch layers. The major overlay contributor that may require additional attention is the Image Placement Error (IPE). When the inter-layer overlay is considered, controlling the intra-field overlay contribution gets more complicated. In addition to the IPE contribution, the TWINSCANTM lens fingerprint in combination with the exposure settings is going to play a role as well. Generally speaking, two subsequent functional layers have different exposure settings. This results in a (non-reticle) additional overlay contribution. In this paper, we have studied the wafer overlay correction capability by RegC® in addition to the TWINSCANTM intrafield corrections to improve the on product overlay performance. RegC® is a reticle intra-volume laser writing technique that causes a predictable deformation element (RegC® deformation element) inside the quartz (Qz) material of a reticle. This technique enables to post-process an existing reticle to correct for instance for IPE. Alternatively, a pre-determined intra-field fingerprint can be added to the reticle such that it results in a straight field after exposure. This second application might be very powerful to correct for instance for (cold) lens fingerprints that cannot be corrected by the scanner itself. Another possible application is the intra-field processing fingerprint. One should realize that a RegC® treatment of a

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.

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.

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.

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.

Genome Editing for Cancer Therapy: Delivery of Cas9 Protein/sgRNA Plasmid via a Gold Nanocluster/Lipid Core-Shell Nanocarrier.

PubMed

Wang, Peng; Zhang, Lingmin; Xie, Yangzhouyun; Wang, Nuoxin; Tang, Rongbing; Zheng, Wenfu; Jiang, Xingyu

2017-11-01

The type II bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (CRISPR-associated protein) system (CRISPR-Cas9) is a powerful toolbox for gene-editing, however, the nonviral delivery of CRISPR-Cas9 to cells or tissues remains a key challenge. This paper reports a strategy to deliver Cas9 protein and single guide RNA (sgRNA) plasmid by a nanocarrier with a core of gold nanoclusters (GNs) and a shell of lipids. By modifying the GNs with HIV-1-transactivator of transcription peptide, the cargo (Cas9/sgRNA) can be delivered into cell nuclei. This strategy is utilized to treat melanoma by designing sgRNA targeting Polo-like kinase-1 ( Plk1 ) of the tumor. The nanoparticle (polyethylene glycol-lipid/GNs/Cas9 protein/sgPlk1 plasmid, LGCP) leads to >70% down-regulation of Plk1 protein expression of A375 cells in vitro. Moreover, the LGCP suppresses melanoma progress by 75% on mice. Thus, this strategy can deliver protein-nucleic acid hybrid agents for gene therapy.

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…

[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.

Cinnamic Acid Analogs as Intervention Catalysts for Overcoming Antifungal Tolerance.

PubMed

Kim, Jong H; Chan, Kathleen L; Cheng, Luisa W

2017-10-21

Disruption of fungal cell wall should be an effective intervention strategy. However, the cell wall-disrupting echinocandin drugs, such as caspofungin (CAS), cannot exterminate filamentous fungal pathogens during treatment. For potency improvement of cell wall-disrupting agents (CAS, octyl gallate (OG)), antifungal efficacy of thirty-three cinnamic acid derivatives was investigated against Saccharomyces cerevisiae slt2 Δ, bck1 Δ, mutants of the mitogen-activated protein kinase (MAPK), and MAPK kinase kinase, respectively, in cell wall integrity system, and glr1 Δ, mutant of CAS-responsive glutathione reductase. Cell wall mutants were highly susceptible to four cinnamic acids (4-chloro-α-methyl-, 4-methoxy-, 4-methyl-, 3-methylcinnamic acids), where 4-chloro-α-methyl- and 4-methylcinnamic acids possessed the highest activity. Structure-activity relationship revealed that 4-methylcinnamic acid, the deoxygenated structure of 4-methoxycinnamic acid, overcame tolerance of glr1 Δ to 4-methoxycinnamic acid, indicating the significance of para substitution of methyl moiety for effective fungal control. The potential of compounds as chemosensitizers (intervention catalysts) to cell wall disruptants (viz., 4-chloro-α-methyl- or 4-methylcinnamic acids + CAS or OG) was assessed according to Clinical Laboratory Standards Institute M38-A. Synergistic chemosensitization greatly lowers minimum inhibitory concentrations of the co-administered drug/agents. 4-Chloro-α-methylcinnamic acid further overcame fludioxonil tolerance of Aspergillus fumigatus antioxidant MAPK mutants ( sakA Δ, mpkC Δ). Collectively, 4-chloro-α-methyl- and 4-methylcinnamic acids possess chemosensitizing capability to augment antifungal efficacy of conventional drug/agents, thus could be developed as target-based (i.e., cell wall disruption) intervention catalysts.

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.

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.

RIFM fragrance ingredient safety assessment, linalyl cinnamate, CAS Registry Number 78-37-5.

PubMed

Api, A M; Belsito, D; Bhatia, S; Bruze, M; Calow, P; Dagli, M L; Dekant, W; Fryer, A D; Kromidas, L; La Cava, S; Lalko, J F; Lapczynski, A; Liebler, D C; Penning, T M; Politano, V T; Ritacco, G; Salvito, D; Schultz, T W; Shen, J; Sipes, I G; Wall, B; Wilcox, D K

2016-11-01

The use of this material under current conditions is supported by existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data show that this material is not genotoxic nor does it have skin sensitization potential. The reproductive and local respiratory toxicity endpoints were completed using the TTC (Threshold of Toxicological Concern) for a Cramer Class I material (0.03 and 1.4 mg/day, respectively). The developmental toxicity endpoint was completed using linalool (CAS # 78-70-6), dehydrolinalool (CAS # 29171-20-8) and cinnamic acid (CAS # 621-82-9) as suitable read across analogs, which provided a MOE > 100. The repeated dose toxicity endpoint was completed using data on the target material which provided a MOE > 100. The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework. Copyright © 2016 Elsevier Ltd. All rights reserved.

CRISPR-Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b.

PubMed

He, Fei; Vestergaard, Gisle; Peng, Wenfang; She, Qunxin; Peng, Xu

2017-02-28

CRISPR-Cas (clustered regularly interspaced short palindromic repeats and the associated genes) constitute adaptive immune systems in bacteria and archaea and they provide sequence specific immunity against foreign nucleic acids. CRISPR-Cas systems are activated by viral infection. However, little is known about how CRISPR-Cas systems are activated in response to viral infection or how their expression is controlled in the absence of viral infection. Here, we demonstrate that both the transcriptional regulator Csa3b, and the type I-A interference complex Cascade, are required to transcriptionally repress the interference gene cassette in the archaeon Sulfolobus. Csa3b binds to two palindromic repeat sites in the promoter region of the cassette and facilitates binding of the Cascade to the promoter region. Upon viral infection, loading of Cascade complexes onto crRNA-matching protospacers leads to relief of the transcriptional repression. Our data demonstrate a mechanism coupling CRISPR-Cas surveillance of protospacers to transcriptional regulation of the interference gene cassette thereby allowing a fast response to viral infection. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

21 CFR 178.3860 - Release agents.

Code of Federal Regulations, 2011 CFR

2011-04-01

...-octadecylcarbamate) (CAS Reg. No. 70892-21-6) produced by the reaction between stoichiometrically equivalent amounts of octadecyl isocyanate and vinyl alcohol/vinyl acetate copolymer; minimum average molecular weight...

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.

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.

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

On the ability of RegCM4 to simulate surface solar radiation patterns over Europe: An assessment using satellite-based observations

NASA Astrophysics Data System (ADS)

Alexandri, Georgia; Georgoulias, Aristeidis K.; Zanis, Prodromos; Tsikerdekis, Athanasios; Katragkou, Eleni; Kourtidis, Konstantinos; Meleti, Charikleia

2015-04-01

We assess here the ability of RegCM4 to simulate the surface solar radiation (SSR) patterns over the European domain. For the needs of this work, a decadal (1999-2009) simulation was implemented at a horizontal resolution of 50km using the first year as a spin-up. The model is driven by emissions from CMIP5 while ERA-interim data were used as lateral boundary conditions. The RegCM4 SSR fields were validated against satellite-based SSR observations from Meteosat First Generation (MFG) and Meteosat Second Generation (MSG) sensors (CM SAF SIS product). The RegCM4 simulations slightly overestimate SSR compared to CM SAF over Europe with the bias being +1.54% in case of MFG (2000-2005) and +3.34% in case of MSG (2006-2009). SSR from RegCM4 is much closer to SSR from CM SAF over land (bias of -1.59% for MFG and +0.66% for MSG) than over ocean (bias of +7.20% for MFG and 8.07% for MSG). In order to understand the reasons of this bias, we proceeded to a detailed assessment of various parameters that define the SSR levels (cloud fractional cover - CFC, cloud optical thickness - COT, cloud droplet effective radius - Re, aerosol optical thickness - AOD, asymmetry factor - ASY, single scattering albedo - SSA, water vapor - WV and surface albedo - ALB). We validated the simulated CFC, COT and Re from RegCM4 against satellite-based observations from MSG and we found that RegCM4 significantly underestimates CFC and Re, and overestimates COT over Europe. The aerosol-related parameters from RegCM4 were compared with values from the aerosol climatology taken into account within CM SAF SSR estimates. AOD is significantly underestimated in our simulations which leads to a positive SSR bias. The RegCM4 WV and ALB were compared with WV values from ERA-interim and ALB climatological observations from CERES which are also taken into account within CM SAF SSR estimates. Finally, with the use of a radiative transfer model (SBDART) we manage to quantify the relative contribution of each of

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…

Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system.

PubMed

Peng, Feng; Wang, Xinyue; Sun, Yang; Dong, Guibin; Yang, Yankun; Liu, Xiuxia; Bai, Zhonghu

2017-11-14

Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum.

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.

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.

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.

21 CFR 74.1328 - D&C Red No. 28.

Code of Federal Regulations, 2012 CFR

2012-04-01

... (CAS Reg. No. 18472-87-2) formed by alkaline hydrolysis of the parent tetrabromotetrachlorofluorescein... sulfates (calculated as sodium salts), not more than 15 percent. Insoluble matter (alkaline solution), not...

21 CFR 74.1328 - D&C Red No. 28.

Code of Federal Regulations, 2014 CFR

2014-04-01

... (CAS Reg. No. 18472-87-2) formed by alkaline hydrolysis of the parent tetrabromotetrachlorofluorescein... sulfates (calculated as sodium salts), not more than 15 percent. Insoluble matter (alkaline solution), not...

21 CFR 74.1328 - D&C Red No. 28.

Code of Federal Regulations, 2010 CFR

2010-04-01

... (CAS Reg. No. 18472-87-2) formed by alkaline hydrolysis of the parent tetrabromotetrachlorofluorescein... sulfates (calculated as sodium salts), not more than 15 percent. Insoluble matter (alkaline solution), not...

21 CFR 74.1328 - D&C Red No. 28.

Code of Federal Regulations, 2013 CFR

2013-04-01

... (CAS Reg. No. 18472-87-2) formed by alkaline hydrolysis of the parent tetrabromotetrachlorofluorescein... sulfates (calculated as sodium salts), not more than 15 percent. Insoluble matter (alkaline solution), not...

[Comparative genomics and evolutionary analysis of CRISPR loci in acetic acid bacteria].

PubMed

Xia, Kai; Liang, Xin-le; Li, Yu-dong

2015-12-01

The clustered regularly interspaced short palindromic repeat (CRISPR) is a widespread adaptive immunity system that exists in most archaea and many bacteria against foreign DNA, such as phages, viruses and plasmids. In general, CRISPR system consists of direct repeat, leader, spacer and CRISPR-associated sequences. Acetic acid bacteria (AAB) play an important role in industrial fermentation of vinegar and bioelectrochemistry. To investigate the polymorphism and evolution pattern of CRISPR loci in acetic acid bacteria, bioinformatic analyses were performed on 48 species from three main genera (Acetobacter, Gluconacetobacter and Gluconobacter) with whole genome sequences available from the NCBI database. The results showed that the CRISPR system existed in 32 species of the 48 strains studied. Most of the CRISPR-Cas system in AAB belonged to type I CRISPR-Cas system (subtype E and C), but type II CRISPR-Cas system which contain cas9 gene was only found in the genus Acetobacter and Gluconacetobacter. The repeat sequences of some CRISPR were highly conserved among species from different genera, and the leader sequences of some CRISPR possessed conservative motif, which was associated with regulated promoters. Moreover, phylogenetic analysis of cas1 demonstrated that they were suitable for classification of species. The conservation of cas1 genes was associated with that of repeat sequences among different strains, suggesting they were subjected to similar functional constraints. Moreover, the number of spacer was positively correlated with the number of prophages and insertion sequences, indicating the acetic acid bacteria were continually invaded by new foreign DNA. The comparative analysis of CRISR loci in acetic acid bacteria provided the basis for investigating the molecular mechanism of different acetic acid tolerance and genome stability in acetic acid bacteria.

Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process

PubMed Central

Li, Ming; Wang, Rui; Zhao, Dahe; Xiang, Hua

2014-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system mediates adaptive immunity against foreign nucleic acids in prokaryotes. However, efficient adaptation of a native CRISPR to purified viruses has only been observed for the type II-A system from a Streptococcus thermophilus industry strain, and rarely reported for laboratory strains. Here, we provide a second native system showing efficient adaptation. Infected by a newly isolated virus HHPV-2, Haloarcula hispanica type I-B CRISPR system acquired spacers discriminatively from viral sequences. Unexpectedly, in addition to Cas1, Cas2 and Cas4, this process also requires Cas3 and at least partial Cascade proteins, which are involved in interference and/or CRISPR RNA maturation. Intriguingly, a preexisting spacer partially matching a viral sequence is also required, and spacer acquisition from upstream and downstream sequences of its target sequence (i.e. priming protospacer) shows different strand bias. These evidences strongly indicate that adaptation in this system strictly requires a priming process. This requirement, if validated also true for other CRISPR systems as implied by our bioinformatic analysis, may help to explain failures to observe efficient adaptation to purified viruses in many laboratory strains, and the discrimination mechanism at the adaptation level that has confused scientists for years. PMID:24265226

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

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

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.

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.

RegA Plays a Key Role in Oxygen-Dependent Establishment of Persistence and in Isocitrate Lyase Activity, a Critical Determinant of In vivo Brucella suis Pathogenicity

PubMed Central

Abdou, Elias; Jiménez de Bagüés, María P.; Martínez-Abadía, Ignacio; Ouahrani-Bettache, Safia; Pantesco, Véronique; Occhialini, Alessandra; Al Dahouk, Sascha; Köhler, Stephan; Jubier-Maurin, Véronique

2017-01-01

For aerobic human pathogens, adaptation to hypoxia is a critical factor for the establishment of persistent infections, as oxygen availability is low inside the host. The two-component system RegB/A of Brucella suis plays a central role in the control of respiratory systems adapted to oxygen deficiency, and in persistence in vivo. Using an original “in vitro model of persistence” consisting in gradual oxygen depletion, we compared transcriptomes and proteomes of wild-type and ΔregA strains to identify the RegA-regulon potentially involved in the set-up of persistence. Consecutive to oxygen consumption resulting in growth arrest, 12% of the genes in B. suis were potentially controlled directly or indirectly by RegA, among which numerous transcriptional regulators were up-regulated. In contrast, genes or proteins involved in envelope biogenesis and in cellular division were repressed, suggesting a possible role for RegA in the set-up of a non-proliferative persistence state. Importantly, the greatest number of the RegA-repressed genes and proteins, including aceA encoding the functional IsoCitrate Lyase (ICL), were involved in energy production. A potential consequence of this RegA impact may be the slowing-down of the central metabolism as B. suis progressively enters into persistence. Moreover, ICL is an essential determinant of pathogenesis and long-term interactions with the host, as demonstrated by the strict dependence of B. suis on ICL activity for multiplication and persistence during in vivo infection. RegA regulates gene or protein expression of all functional groups, which is why RegA is a key regulator of B. suis in adaptation to oxygen depletion. This function may contribute to the constraint of bacterial growth, typical of chronic infection. Oxygen-dependent activation of two-component systems that control persistence regulons, shared by several aerobic human pathogens, has not been studied in Brucella sp. before. This work therefore contributes

21 CFR 184.1230 - Calcium sulfate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium sulfate. 184.1230 Section 184.1230 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DIRECT... GRAS § 184.1230 Calcium sulfate. (a) Calcium sulfate (CaSO4, CAS Reg. No. 7778-18-9 or CaSO4·2H2O, CAS...

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

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.

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.

21 CFR 173.170 - Aminoglycoside 3′-phospho-trans-ferase II.

Code of Federal Regulations, 2014 CFR

2014-04-01

... SERVICES (CONTINUED) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme... is the enzyme aminoglycoside 3′-phosphotransferase II (CAS Reg. No. 58943-39-8) which catalyzes the...

21 CFR 173.170 - Aminoglycoside 3′-phospho-trans-ferase II.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.170 Aminoglycoside 3′-phospho-trans... prescribed conditions: (a) The food additive is the enzyme aminoglycoside 3′-phosphotransferase II (CAS Reg...

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

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.

Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands.

PubMed

Vercoe, Reuben B; Chang, James T; Dy, Ron L; Taylor, Corinda; Gristwood, Tamzin; Clulow, James S; Richter, Corinna; Przybilski, Rita; Pitman, Andrew R; Fineran, Peter C

2013-04-01

In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas-mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA-targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.

On the ability of RegCM4 regional climate model to simulate surface solar radiation patterns over Europe: an assessment using satellite-based observations

NASA Astrophysics Data System (ADS)

Alexandri, G.; Georgoulias, A. K.; Zanis, P.; Katragkou, E.; Tsikerdekis, A.; Kourtidis, K.; Meleti, C.

2015-07-01

In this work, we assess the ability of RegCM4 regional climate model to simulate surface solar radiation (SSR) patterns over Europe. A decadal RegCM4 run (2000-2009) was implemented and evaluated against satellite-based observations from the Satellite Application Facility on Climate Monitoring (CM SAF) showing that the model simulates adequately the SSR patterns over the region. The bias between RegCM4 and CM SAF is +1.54 % for MFG (Meteosat First Generation) and +3.34 % for MSG (Meteosat Second Generation) observations. The relative contribution of parameters that determine the transmission of solar radiation within the atmosphere to the deviation appearing between RegCM4 and CM SAF SSR is also examined. Cloud macrophysical and microphysical properties such as cloud fractional cover (CFC), cloud optical thickness (COT) and cloud effective radius (Re) from RegCM4 are evaluated against data from CM SAF. The same procedure is repeated for aerosol optical properties such as aerosol optical depth (AOD), asymmetry factor (ASY) and single scattering albedo (SSA), as well as other parameters including surface broadband albedo (ALB) and water vapor amount (WV) using data from MACv1 aerosol climatology, from CERES satellite sensors and from ERA-Interim reanalysis. It is shown here that the good agreement between RegCM4 and satellite-based SSR observations can be partially attributed to counteracting effects among the above mentioned parameters. The contribution of each parameter to the RegCM4-CM SAF SSR deviations is estimated with the combined use of the aforementioned data and a radiative transfer model (SBDART). CFC, COT and AOD are the major determinants of these deviations; however, the other parameters also play an important role for specific regions and seasons.

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

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

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

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).

Rainfall forecast in the Upper Mahaweli basin in Sri Lanka using RegCM model

NASA Astrophysics Data System (ADS)

Muhammadh, K. M.; Mafas, M. M. M.; Weerakoon, S. B.

2017-04-01

The Upper Mahaweli basin is the upper most sub basin of 788 km2 in size above Polgolla barrage in the Mahaweli River, the longest river in Sri Lanka which starts from the central hills of the island and drains to the sea at the North-east coast. Rainfall forecast in the Upper Mahaweli basin is important for issuing flood warning in the river downstream of the reservoirs, landslide warning in the settlements in hilly areas. Anticipatory water management in the basin including reservoir operations, barrage gate operation for releasing water for irrigation and flood control also require reliable rainfall and runoff prediction in the sub basin. In this study, the Regional Climate Model (RegCM V4.4.5.11) is calibrated for the basin to dynamically downscale reanalysis weather data of Global Climate Model (GCM) to forecast the rainfall in the basin. Observed rainfalls at gauging stations within the basin were used for model calibration and validation. The observed rainfall data was analysed using ARC GIS and the output of RegCM was analysed using GrADS tool. The output of the model and the observed precipitation were obtained on grids of size 0.1 degrees and the accuracy of the predictions were analysed using RMSE and Mean Model Absolute Error percentage (MAME %). The predictions by the calibrated RegCM model for the basin is shown to be satisfactory. The model is a useful tool for rainfall forecast in the Upper Mahaweli River basin.

21 CFR 173.170 - Aminoglycoside 3′-phospho-trans-ferase II.

Code of Federal Regulations, 2013 CFR

2013-04-01

... FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.170 Aminoglycoside 3′-phospho... prescribed conditions: (a) The food additive is the enzyme aminoglycoside 3′-phosphotransferase II (CAS Reg...

21 CFR 173.170 - Aminoglycoside 3′-phospho-trans-ferase II.

Code of Federal Regulations, 2012 CFR

2012-04-01

... FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.170 Aminoglycoside 3′-phospho... prescribed conditions: (a) The food additive is the enzyme aminoglycoside 3′-phosphotransferase II (CAS Reg...

21 CFR 173.170 - Aminoglycoside 3′-phospho-trans-ferase II.

Code of Federal Regulations, 2011 CFR

2011-04-01

... FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.170 Aminoglycoside 3′-phospho... prescribed conditions: (a) The food additive is the enzyme aminoglycoside 3′-phosphotransferase II (CAS Reg...

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.

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

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

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.

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

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.

21 CFR 177.2450 - Polyamide-imide resins.

Code of Federal Regulations, 2013 CFR

2013-04-01

...) For the purpose of this section the polyamide-imide resins are derived from the condensation reaction...) The polyamide-imide resins (CAS Reg. No. 31957-38-7) derived from the condensation reaction of...

21 CFR 177.2450 - Polyamide-imide resins.

Code of Federal Regulations, 2012 CFR

2012-04-01

...) For the purpose of this section the polyamide-imide resins are derived from the condensation reaction...) The polyamide-imide resins (CAS Reg. No. 31957-38-7) derived from the condensation reaction of...

40 CFR 180.920 - Inert ingredients used pre-harvest; exemptions from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Cucurbitacin) Gustatory stimulant Butyl stearate Defoamer γ-Butyrolactone Solvent C.I. Pigment Blue #15 (CAS Reg. No. 147-14-8; containing no more than 50 ppm polychlorinated biphenyls (PCBs)) For seed treament...

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

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.

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.

The regulation of K- and L-cell activity by GLUT2 and the calcium-sensing receptor CasR in rat small intestine

PubMed Central

Mace, Oliver J; Schindler, Marcus; Patel, Sonal

2012-01-01

Intestinal enteroendocrine cells (IECs) secrete gut peptides in response to both nutrients and non-nutrients. Glucose and amino acids both stimulate gut peptide secretion. Our hypothesis was that the facilitative glucose transporter, GLUT2, could act as a glucose sensor and the calcium-sensing receptor, CasR, could detect amino acids in the intestine to modify gut peptide secretion. We used isolated loops of rat small intestine to study the secretion of gluco-insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) secretion stimulated by luminal perfusion of nutrients or bile acid. Inhibition of the sodium-dependent glucose cotransporter 1 (SGLT1) with phloridzin partially inhibited GIP, GLP-1 and PYY secretion by 45%, suggesting another glucose sensor might be involved in modulating peptide secretion. The response was completely abolished in the presence of the GLUT2 inhibitors phloretin or cytochalasin B. Given that GLUT2 modified gut peptide secretion stimulated by glucose, we investigated whether it was involved in the secretion of gut peptide by other gut peptide secretagogues. Phloretin completely abolished gut peptide secretion stimulated by artificial sweetener (sucralose), dipeptide (glycylsarcosine), lipid (oleoylethanolamine), short chain fatty acid (propionate) and major rat bile acid (taurocholate) indicating a fundamental position for GLUT2 in the gut peptide secretory mechanism. We investigated how GLUT2 was able to influence gut peptide secretion mediated by a diverse range of stimulators and discovered that GLUT2 affected membrane depolarisation through the closure of K+ATP-sensitive channels. In the absence of SGLT1 activity (or presence of phloridzin), the secretion of GIP, GLP-1 and PYY was sensitive to K+ATP-sensitive channel modulators tolbutamide and diazoxide. l-Amino acids phenylalanine (Phe), tryptophan (Trp), asparagine (Asn), arginine (Arg) and glutamine (Gln) also stimulated GIP, GLP-1 and PYY

Extratropical Cyclones over Southwestern Atlantic Ocean: Present and Future Climates projected by RegCM4

NASA Astrophysics Data System (ADS)

Reboita, Michelle; Rodrigues, Marcelo; da Rocha, Rosmeri

2017-04-01

This study shows some of the climatological features of the extratropical cyclones in present and future climate over Southwestern Atlantic Ocean (SAO). The projections were carried out with Regional Climate Model (RegCM4) nested in HadGEM2-ES global model outputs and using representative concentration pathway 8.5 (RCP8.5) from the CMIP5. The simulations considered the South America domain suggested by CORDEX, horizontal grid spacing of 50 km, 18 sigma-pressure levels in the vertical. An objective tracking scheme based on cyclonic relative vorticity calculated using the wind at 925 hPa was used to identify the cyclones. All cyclones with relative vorticity lower than the -1.5 x 10-5 s-1 and with lifetime higher or equal 24 hours were included in the climatology. Considering the period from 1979 to 2098, RegCM4 and HadGEM2-ES project a negative trend in the frequency of the extratropical cyclones over SAO, with the biggest negative trend occuring in the latitudinal band between 40°S and 57.5°S. This result can be associated with the southward displacement of the baroclinic zone which contributes to the cyclones move to south leaving the region analyzed. The three subregions with largest cyclogenetic activity discussed in the literature (southeast coast of Brazil - RG1, coast of Uruguay and southern Brazil - RG2; east coast of Argentina - RG3) were better reproduced in RegCM4 than in HadGEM2-ES. Therefore, RegCM4 downscaling ads value in the HadGEM2-ES projections. The frequency of cyclones in present (1979-2005) and future climate (2070-2098) is higher in winter and lower in summer. Regarding the mean characteristics of the cyclones (life time, travel distance, velocity, initial relative vorticity and total average vorticity), both models successfully reproduced those obtained in the reanalysis (NCEP1, NCEP2, CFSR, ERA40 and ERA-Interim) and there are no significant differences in the future climate compared with the present.

Simulation and spatiotemporal pattern of air temperature and precipitation in Eastern Central Asia using RegCM.

PubMed

Meng, Xianyong; Long, Aihua; Wu, Yiping; Yin, Gang; Wang, Hao; Ji, Xiaonan

2018-02-26

Central Asia is a region that has a large land mass, yet meteorological stations in this area are relatively scarce. To address this data issues, in this study, we selected two reanalysis datasets (the ERA40 and NCEP/NCAR) and downscaled them to 40 × 40 km using RegCM. Then three gridded datasets (the CRU, APHRO, and WM) that were extrapolated from the observations of Central Asian meteorological stations to evaluate the performance of RegCM and analyze the spatiotemporal distribution of precipitation and air temperature. We found that since the 1960s, the air temperature in Xinjiang shows an increasing trend and the distribution of precipitation in the Tianshan area is quite complex. The precipitation is increasing in the south of the Tianshan Mountains (Southern Xinjiang, SX) and decreasing in the mountainous areas. The CRU and WM data indicate that precipitation in the north of the Tianshan Mountains (Northern Xinjiang, NX) is increasing, while the APHRO data show an opposite trend. The downscaled results from RegCM are generally consistent with the extrapolated gridded datasets in terms of the spatiotemporal patterns. We believe that our results can provide useful information in developing a regional climate model in Central Asia where meteorological stations are scarce.

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.

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.

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...

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.

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.

Optimization of genome editing through CRISPR-Cas9 engineering