Science.gov

Sample records for series temporelles cas

  1. Delivery of Cas9 Protein into Mouse Zygotes through a Series of Electroporation Dramatically Increases the Efficiency of Model Creation.

    PubMed

    Wang, Wenbo; Kutny, Peter M; Byers, Shannon L; Longstaff, Charles J; DaCosta, Michael J; Pang, Changhong; Zhang, Yingfan; Taft, Robert A; Buaas, Frank W; Wang, Haoyi

    2016-05-20

    Previously we established Zygote Electroporation of Nucleases (ZEN) technology as an efficient and high-throughput way to generate genetically modified mouse models. However, there were significant variations of the targeting efficiency among different genomic loci using our previously published protocol. In this study, we improved the ZEN technology by delivering Cas9 protein into mouse zygotes through a series of electroporation. Using this approach, we were able to introduce precise nucleotide substitutions, large segment deletion and short segment insertion into targeted loci with high efficiency. PMID:27210041

  2. [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…

  3. Cas9 Functionally Opens Chromatin.

    PubMed

    Barkal, Amira A; Srinivasan, Sharanya; Hashimoto, Tatsunori; Gifford, David K; Sherwood, Richard I

    2016-01-01

    Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding. PMID:27031353

  4. Cas9 Functionally Opens Chromatin

    PubMed Central

    Barkal, Amira A.; Srinivasan, Sharanya; Hashimoto, Tatsunori; Gifford, David K.; Sherwood, Richard I.

    2016-01-01

    Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding. PMID:27031353

  5. Prediction des vibrations eoliennes d'un systeme conducteur-amortisseur avec une methode temporelle non lineaire

    NASA Astrophysics Data System (ADS)

    Langlois, Sebastien

    Les vibrations eoliennes sont la cause principale de bris de conducteurs en fatigue des lignes aeriennes de transport d'energie electrique. Ces vibrations sont dues a des detachements tourbillonnaires produits dans le sillage du conducteur. Une methode commune de reduction des vibrations est l'ajout d'amortisseurs de vibrations pres des pinces de suspension. Contrairement aux essais en ligne experimentale, la modelisation numerique permet d'evaluer rapidement et a faible cout la performance d'un amortisseur de vibration sur une portee de ligne aerienne. La technologie la plus frequemment utilisee fait appel au principe de balance d'energie (PBE) en evaluant le niveau de vibrations pour lequel la puissance injectee par le vent est egale a la puissance dissipee par le conducteur et l'amortisseur. Les methodes actuelles pour la prediction des vibrations reposent sur des hypotheses simplificatrices quant a la modelisation de l'interaction conducteur-amortisseur. Une approche prometteuse pour la prediction des vibrations est l'utilisation d'un modele numerique temporel non lineaire qui permet de mieux representer la masse, la geometrie, la rigidite et l'amortissement du systeme. L'objectif principal de ce projet de recherche est de developper un modele numerique avec integration temporelle directe d'un conducteur et d'un amortisseur en vibration permettant de reproduire le comportement dynamique du systeme pour la gamme de frequence et d'amplitude typique des vibrations eoliennes des conducteurs. Un modele par elements finis d'un conducteur seul en vibration resolu par integration temporelle directe a d'abord ete developpe en considerant une rigidite de flexion variable. Comme une rigidite de flexion constante et egale a 50% de la rigidite de flexion maximale theorique ( EImax) est jugee adequate pour la modelisation du conducteur, c'est cette valeur qui a ete utilisee pour la suite du projet. Ensuite, des modeles non-lineaires pour deux types d'amortisseur de

  6. Dual nuclease activity of a Cas2 protein in CRISPR-Cas subtype I-B of Leptospira interrogans.

    PubMed

    Dixit, Bhuvan; Ghosh, Karukriti Kaushik; Fernandes, Gary; Kumar, Pankaj; Gogoi, Prerana; Kumar, Manish

    2016-04-01

    Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 carries a set of cas genes associated with CRISPR-Cas subtype I-B. Herein, we report for the first time active transcription of a set of cas genes (cas1 to cas8) of L. interrogans where cas4, cas1, cas2 and cas6, cas3, cas8, cas7, cas5 are clustered together in two independent operons. As an initial step toward comprehensive understanding of CRISPR-Cas system in spirochete, the biochemical study of one of the core Leptospira Cas2 proteins (Lep_Cas2) showed nuclease activity on both DNA and RNA in a nonspecific manner. Additionally, unlike other known Cas2 proteins, Lep_Cas2 showed metal-independent RNase activity and preferential activity on RNA over DNA. These results provide insight for understanding Cas2 diversity existing in the prokaryotic adaptive immune system. PMID:26950513

  7. 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

  8. 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…

  9. "CAS" Statement of Shared Ethical Principles

    ERIC Educational Resources Information Center

    Council for the Advancement of Standards in Higher Education, 2006

    2006-01-01

    The Council for the Advancement of Standards in Higher Education (CAS) has served as a voice for quality assurance and promulgation of standards in higher education for over 25 years. CAS was established to promote inter-association efforts to address quality assurance, student learning, and professional integrity. CAS includes membership of over…

  10. CAS-1 lunar soil simulant

    NASA Astrophysics Data System (ADS)

    Zheng, Yongchun; Wang, Shijie; Ouyang, Ziyuan; Zou, Yongliao; Liu, Jianzhong; Li, Chunlai; Li, Xiongyao; Feng, Junming

    2009-02-01

    Lunar soil simulant is a geochemical reproduction of lunar regolith, and is needed for lunar science and engineering researches. This paper describes a new lunar soil simulant, CAS-1, prepared by the Chinese Academy of Sciences, to support lunar orbiter, soft-landing mission and sample return missions of China’s Lunar Exploration Program, which is scheduled for 2004 2020. Such simulants should match the samples returned from the Moon, all collected from the lunar regolith rather than outcrops. The average mineral and chemical composition of lunar soil sample returned from the Apollo 14 mission, which landed on the Fra Mauro Formation, is chosen as the model for the CAS-1 simulant. Source material for this simulant was a low-Ti basaltic scoria dated at 1600 years from the late Quaternary volcanic area in the Changbai Mountains of northeast China. The main minerals of this rock are pyroxene, olivine, and minor plagioclase, and about 20 40% modal glass. The scoria was analyzed by XRF and found to be chemically similar to Apollo 14 lunar sample 14163. It was crushed in an impact mill with a resulting median particle size 85.9 μm, similar to Apollo soils. Bulk density, shear resistance, complex permittivity, and reflectance spectra were also similar to Apollo 14 soil. We conclude that CAS-1 is an ideal lunar soil simulant for science and engineering research of future lunar exploration program.

  11. Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9

    PubMed Central

    Feng, Yan; Chen, Cheng; Han, Yuxiang; Chen, Zelin; Lu, Xiaochan; Liang, Fang; Li, Song; Qin, Wei; Lin, Shuo

    2016-01-01

    The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5′-NGG-3′ protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology. PMID:27317783

  12. Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9.

    PubMed

    Feng, Yan; Chen, Cheng; Han, Yuxiang; Chen, Zelin; Lu, Xiaochan; Liang, Fang; Li, Song; Qin, Wei; Lin, Shuo

    2016-01-01

    The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5'-NGG-3' protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology. PMID:27317783

  13. Caractérisation spectrale et temporelle de l'émission X issue de l'interaction laser-agrégats

    NASA Astrophysics Data System (ADS)

    Bonté, C.; Fourment, C.; Harmand, M.; Jouin, H.; Micheau, S.; Peyrusse, O.; Pons, B.; Santos, J. J.

    2006-12-01

    Les agrégats de gaz rares constituent un état de la matière intermédiaire entre les cibles solides massives et les atomes en phase gazeuse. Il a été démontré que les agrégats irradiés sont sources d'ions, d'électrons, de neutrons ainsi que de rayonnement allant du visible aux X durs. Cette source peut-être produite avec un taux de répétition élevé et a l'avantage de ne pas produire de débris, dommageables pour les optiques notamment, et de présenter une très forte conversion de l'énergie laser incidente. Nous nous intéressons au rayonnement X particulièrement, en le caractérisant en intensité, spectre et durée, comme préalable à toute application de cette source X et comme moyen privilégié d'étude de la physique des plasmas nanométriques chauds et denses. En collaboration avec l'INRS-Énergie (Varenne, Qc, Canada), nous avons mis en œuvre une caméra à balayage de fente dont la résolution temporelle est de 800 fs rms. En focalisant des impulsions laser courtes (30 fs 5 ps) et intenses (jusqu'à 1017 W/cm2) sur des agrégats d'argon dont le rayon varie de 15 à 30 nm, nous avons démontré que l'émission X dont l'énergie est supérieure à 2 keV est plus courte que 2 ps, limité par la résolution temporelle. En couplant la caméra à un cristal tronconique, dont la conception a été réalisée au LULI (Palaiseau, France), nous nous sommes intéressés au rayonnement de couche K dans la gamme 2,9 - 3,2 keV. Nous avons démontré que ce rayonnement a une durée inférieure à 3 ps (limite de la résolution temporelle), et que les raies étaient émises avec un écart relatif inférieur à 1 ps. Une simulation basée sur le modèle nano-plasma proposé par T. Ditmire et sur le code collisionnel-radiatif Transpec a été développée au CELIA. Les spectres X résolus en temps calculés reproduisent à la fois la brièveté d'émission du rayonnement X et les états de charge élevés observés.

  14. CAS as Environments for Implementing Mathematical Microworlds.

    ERIC Educational Resources Information Center

    Alpers, Burkhard

    2002-01-01

    Investigates whether computer algebra systems (CAS) are suitable environments for implementing mathematical microworlds. Recalls what constitutes a microworld and explores how CAS can be used for implementation, stating potentials as well as limitations. Provides as an example the microworld "Formula 1", implemented in Maple Software. (Author/KHR)

  15. CAS

    SciTech Connect

    Martinez, B.; Pomeroy, G. )

    1989-12-02

    The Security Alarm System is a data acquisition and control system which collects data from intrusion sensors and displays the information in a real-time environment for operators. The Access Control System monitors and controls the movement of personnel with the use of card readers and biometrics hand readers.

  16. RXTE Observations of Cas A

    NASA Technical Reports Server (NTRS)

    Rothschild, R. E.; Lingenfelter, R. E.; Heindl, W. A.; Blanco, P. R.; Pelling, M. R.; Gruber, D. E.; Allen, G. E.; Jahoda, K.; Swank, J. H.; Woosley, S. E.; Nomoto, K.; Higdon, J. C.; Dermer, Charles D. (Editor); Strickman, Mark S. (Editor); Kurfess, James D. (Editor)

    1997-01-01

    The exciting detection by the COMPTEL instrument of the 1157 keV Ti-44 line from the supernova remnant Cas A sets important new constraints on supernova dynamics and nucleosynthesis. The Ti-44 decay also produces x-ray lines at 68 and 78 keV, whose flux should be essentially the same as that of the gamma ray line. The revised COMPTEL flux of 4 x l0(exp -5) cm(exp -2)s(exp -1) is very near the sensitivity limit for line detection by the HEXTE instrument on RXTE. We report on the results from two RXTE observations - 20 ks during In Orbit Checkout in January 1996 and 200 ks in April 1996. We also find a strong continuum emission suggesting cosmic ray electron acceleration in the remnant.

  17. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 7 2014-10-01 2014-10-01 false Types of CAS coverage... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  18. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Types of CAS coverage... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  19. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 7 2013-10-01 2012-10-01 true Types of CAS coverage. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  20. 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…

  1. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 7 2012-10-01 2012-10-01 false Types of CAS coverage... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. V723 Cas a borderline classical nova

    NASA Astrophysics Data System (ADS)

    Friedjung, M.; Iijima, T.

    2002-11-01

    V723 Cas had a light curve similar to that of HR Del before maximum, with a very slow pre-maximum rise, explained according to [2] by the presence of an optically thin wind before maximum unlike the optically thick wind generally seen for classical novae after maximum. Examination of the Fe II emission lines by the SAC method, is compatible with this also having been the case for V723 Cas.

  4. Characterization of Cas9-Guide RNA Orthologs.

    PubMed

    Braff, Jonathan L; Yaung, Stephanie J; Esvelt, Kevin M; Church, George M

    2016-01-01

    In light of the multitude of new Cas9-mediated functionalities, the ability to carry out multiple Cas9-enabled processes simultaneously and in a single cell is becoming increasingly valuable. Accomplishing this aim requires a set of Cas9-guide RNA (gRNA) pairings that are functionally independent and insulated from one another. For instance, two such protein-gRNA complexes would allow for concurrent activation and editing at independent target sites in the same cell. The problem of establishing orthogonal CRISPR systems can be decomposed into three stages. First, putatively orthogonal systems must be identified with an emphasis on minimizing sequence similarity of the Cas9 protein and its associated RNAs. Second, the systems must be characterized well enough to effectively express and target the systems using gRNAs. Third, the systems should be established as orthogonal to one another by testing for activity and cross talk. Here, we describe the value of these orthogonal CRISPR systems, outline steps for selecting and characterizing potentially orthogonal Cas9-gRNA pairs, and discuss considerations for the desired specificity in Cas9-coupled functions. PMID:27140923

  5. Putting the CAS Standards to Work. Training Manual for the CAS Self Assessment Guides.

    ERIC Educational Resources Information Center

    Yerian, Jean M.; Miller, Theodore K., Ed.

    These 18 self-assessment guides and training manual from the Council for the Advancement of Standards (CAS) for Student Services/Development Programs translate the CAS Standards and Guidelines of 1986 into a format for self-study purposes. These self-study guides allow an institution to assure compliance with minimally-acceptable practice, gain an…

  6. Exploring Fourier Series and Gibbs Phenomenon Using Mathematica

    ERIC Educational Resources Information Center

    Ghosh, Jonaki B.

    2011-01-01

    This article describes a laboratory module on Fourier series and Gibbs phenomenon which was undertaken by 32 Year 12 students. It shows how the use of CAS played the role of an "amplifier" by making higher level mathematical concepts accessible to students of year 12. Using Mathematica students were able to visualise Fourier series of functions…

  7. CAS2D- NONROTATING BLADE-TO-BLADE, STEADY, POTENTIAL TRANSONIC CASCADE FLOW ANALYSIS CODE

    NASA Technical Reports Server (NTRS)

    Dulikravich, D. S.

    1994-01-01

    An exact, full-potential-equation model for the steady, irrotational, homoentropic, and homoenergetic flow of a compressible, inviscid fluid through a two-dimensional planar cascade together with its appropriate boundary conditions has been derived. The CAS2D computer program numerically solves an artificially time-dependent form of the actual full-potential-equation, providing a nonrotating blade-to-blade, steady, potential transonic cascade flow analysis code. Comparisons of results with test data and theoretical solutions indicate very good agreement. In CAS2D, the governing equation is discretized by using type-dependent, rotated finite differencing and the finite area technique. The flow field is discretized by providing a boundary-fitted, nonuniform computational mesh. This mesh is generated by using a sequence of conformal mapping, nonorthogonal coordinate stretching, and local, isoparametric, bilinear mapping functions. The discretized form of the full-potential equation is solved iteratively by using successive line over relaxation. Possible isentropic shocks are captured by the explicit addition of an artificial viscosity in a conservative form. In addition, a four-level, consecutive, mesh refinement feature makes CAS2D a reliable and fast algorithm for the analysis of transonic, two-dimensional cascade flows. The results from CAS2D are not directly applicable to three-dimensional, potential, rotating flows through a cascade of blades because CAS2D does not consider the effects of the Coriolis force that would be present in the three-dimensional case. This program is written in FORTRAN IV for batch execution and has been implemented on an IBM 370 series computer with a central memory requirement of approximately 200K of 8 bit bytes. The CAS2D program was developed in 1980.

  8. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  9. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  10. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  11. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  12. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  13. CRISPR-Cas: Revolutionising genome engineering.

    PubMed

    Nicholson, Samantha Anne; Pepper, Michael Sean

    2016-09-01

    The ability to permanently alter or repair the human genome has been the subject of a number of science fiction films, but with the recent advent of several customisable sequence-specific endonuclease technologies, genome engineering looks set to become a clinical reality in the near future. This article discusses recent advancements in the technology called 'clustered regularly interspaced palindromic repeat (CRISPR)-associated genes' (CRISPR-Cas), the potential of CRISPR-Cas to revolutionise molecular medicine, and the ethical and regulatory hurdles facing its application. PMID:27601107

  14. Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

    PubMed

    Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Topkar, Ved V; Nguyen, Nhu T; Zheng, Zongli; Gonzales, Andrew P W; Li, Zhuyun; Peterson, Randall T; Yeh, Jing-Ruey Joanna; Aryee, Martin J; Joung, J Keith

    2015-07-23

    Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities. PMID:26098369

  15. Engineered CRISPR-Cas9 nucleases with altered PAM specificities

    PubMed Central

    Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.; Topkar, Ved; Nguyen, Nhu T.; Zheng, Zongli; Gonzales, Andrew P.W.; Li, Zhuyun; Peterson, Randall T.; Yeh, Jing-Ruey Joanna; Aryee, Martin J.; Joung, J. Keith

    2015-01-01

    Although CRISPR-Cas9 nucleases are widely used for genome editing1, 2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3–6. As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-Seq analysis7. In addition, we identified and characterized another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also found that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities. PMID:26098369

  16. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2012-10-01 2012-10-01 false CAS applicability....

  17. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2014-10-01 2014-10-01 false Types of CAS coverage....

  18. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false CAS applicability....

  19. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2012-10-01 2012-10-01 false Types of CAS coverage....

  20. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 7 2012-10-01 2012-10-01 false CAS applicability. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  1. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false Types of CAS coverage....

  2. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false CAS applicability. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  3. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false Types of CAS coverage....

  4. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false CAS applicability....

  5. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 7 2014-10-01 2014-10-01 false CAS applicability. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  6. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2014-10-01 2014-10-01 false CAS applicability....

  7. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 7 2013-10-01 2012-10-01 true CAS applicability. 9903.201... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  8. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Types of CAS coverage....

  9. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false CAS applicability....

  10. Structural plasticity and in vivo activity of Cas1 from the type I-F CRISPR-Cas system.

    PubMed

    Wilkinson, Max E; Nakatani, Yoshio; Staals, Raymond H J; Kieper, Sebastian N; Opel-Reading, Helen K; McKenzie, Rebecca E; Fineran, Peter C; Krause, Kurt L

    2016-04-15

    CRISPR-Cas systems are adaptive immune systems in prokaryotes that provide protection against viruses and other foreign DNA. In the adaptation stage, foreign DNA is integrated into CRISPR (clustered regularly interspaced short palindromic repeat) arrays as new spacers. These spacers are used in the interference stage to guide effector CRISPR associated (Cas) protein(s) to target complementary foreign invading DNA. Cas1 is the integrase enzyme that is central to the catalysis of spacer integration. There are many diverse types of CRISPR-Cas systems, including type I-F systems, which are typified by a unique Cas1-Cas2-3 adaptation complex. In the present study we characterize the Cas1 protein of the potato phytopathogen Pectobacterium atrosepticum, an important model organism for understanding spacer acquisition in type I-F CRISPR-Cas systems. We demonstrate by mutagenesis that Cas1 is essential for adaptation in vivo and requires a conserved aspartic acid residue. By X-ray crystallography, we show that although P. atrosepticum Cas1 adopts a fold conserved among other Cas1 proteins, it possesses remarkable asymmetry as a result of structural plasticity. In particular, we resolve for the first time a flexible, asymmetric loop that may be unique to type I-F Cas1 proteins, and we discuss the implications of these structural features for DNA binding and enzymatic activity. PMID:26929403

  11. Historic light curve of V890 Cas

    NASA Astrophysics Data System (ADS)

    Nesci, R.

    2016-05-01

    The variability of V890 Cas is studied with 87 -band plates of the Asiago archive. The star shows variations of about 5 mag with an average magnitude =13 and a period of 486 days. An 5.0 color index is also derived near the maximum luminosity.

  12. Using the CAS Standards in Assessment Projects

    ERIC Educational Resources Information Center

    Dean, Laura A.

    2013-01-01

    This chapter provides an overview of the use of professional standards of practice in assessment and of the Council for the Advancement of Standards in Higher Education (CAS). It outlines a model for conducting program self-studies and discusses the importance of implementing change based on assessment results.

  13. Lessons Learned on Management of CAS Development.

    ERIC Educational Resources Information Center

    Boyadjieff, Kiril

    1995-01-01

    Computer-assisted studies (CAS) attract foreign language professionals' attention due to the reliability of personal computers, the decreasing cost of available technology, and the new generation of students for whom electronic media are a familiar habitat. This article focuses on a project of the Defense Language Institute that produced over…

  14. CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA.

    PubMed

    Zhou, Jianting; Wu, Ronghai; Xue, Xiaoli; Qin, Zhongjun

    2016-08-19

    Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated 'CasHRA (Cas9-facilitated Homologous Recombination Assembly)' that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 (Minimal Genome of Escherichia coli) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions. PMID:27220470

  15. Controlling UCAVs by JTACs in CAS missions

    NASA Astrophysics Data System (ADS)

    Kumaş, A. E.

    2014-06-01

    By means of evolving technology, capabilities of UAVs (Unmanned Aerial Vehicle)s are increasing rapidly. This development provides UAVs to be used in many different areas. One of these areas is CAS (Close Air Support) mission. UAVs have several advantages compared to manned aircraft, however there are also some problematic areas. The remote controlling of these vehicles from thousands of nautical miles away via satellite may lead to various problems both ethical and tactical aspects. Therefore, CAS missions require a good level of ALI (Air-Land Integration), a high SA (situational awareness) and precision engagement. In fact, there is an aware friendly element in the target area in CAS missions, unlike the other UAV operations. This element is an Airman called JTAC (Joint Terminal Attack Controller). Unlike the JTAC, UAV operators are too far away from target area and use the limited FOV (Field of View) provided by camera and some other sensor data. In this study, target area situational awareness of a UAV operator and a JTAC, in a high-risk mission for friendly ground forces and civilians such as CAS, are compared. As a result of this comparison, answer to the question who should control the UCAV (Unmanned Combat Aerial Vehicle) in which circumstances is sought. A literature review is made in UAV and CAS fields and recent air operations are examined. The control of UCAV by the JTAC is assessed by SWOT analysis and as a result it is deduced that both control methods can be used in different situations within the framework of the ROE (Rules Of Engagement) is reached.

  16. 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. PMID:26875867

  17. 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

  18. In vivo genome editing using Staphylococcus aureus Cas9

    PubMed Central

    Ran, F. Ann; Cong, Le; Yan, Winston X.; Scott, David A.; Gootenberg, Jonathan S.; Kriz, Andrea J.; Zetsche, Bernd; Shalem, Ophir; Wu, Xuebing; Makarova, Kira S.; Koonin, Eugene; Sharp, Phillip A.; Zhang, Feng

    2015-01-01

    The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that employ the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologs and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being >1kb shorter. We packaged SaCas9 and its sgRNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further demonstrate the power of using BLESS to assess the genome-wide targeting specificity of SaCas9 and SpCas9, and show that SaCas9 can mediate genome editing in vivo with high specificity. PMID:25830891

  19. Photoactivatable CRISPR-Cas9 for optogenetic genome editing.

    PubMed

    Nihongaki, Yuta; Kawano, Fuun; Nakajima, Takahiro; Sato, Moritoshi

    2015-07-01

    We describe an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR-Cas9 genome editing in human cells. paCas9 consists of split Cas9 fragments and photoinducible dimerization domains named Magnets. In response to blue light irradiation, paCas9 expressed in human embryonic kidney 293T cells induces targeted genome sequence modifications through both nonhomologous end joining and homology-directed repair pathways. Genome editing activity can be switched off simply by extinguishing the light. We also demonstrate activation of paCas9 in spatial patterns determined by the sites of irradiation. Optogenetic control of targeted genome editing should facilitate improved understanding of complex gene networks and could prove useful in biomedical applications. PMID:26076431

  20. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems

    PubMed Central

    2011-01-01

    Background The CRISPR-Cas adaptive immunity systems that are present in most Archaea and many Bacteria function by incorporating fragments of alien genomes into specific genomic loci, transcribing the inserts and using the transcripts as guide RNAs to destroy the genome of the cognate virus or plasmid. This RNA interference-like immune response is mediated by numerous, diverse and rapidly evolving Cas (CRISPR-associated) proteins, several of which form the Cascade complex involved in the processing of CRISPR transcripts and cleavage of the target DNA. Comparative analysis of the Cas protein sequences and structures led to the classification of the CRISPR-Cas systems into three Types (I, II and III). Results A detailed comparison of the available sequences and structures of Cas proteins revealed several unnoticed homologous relationships. The Repeat-Associated Mysterious Proteins (RAMPs) containing a distinct form of the RNA Recognition Motif (RRM) domain, which are major components of the CRISPR-Cas systems, were classified into three large groups, Cas5, Cas6 and Cas7. Each of these groups includes many previously uncharacterized proteins now shown to adopt the RAMP structure. Evidence is presented that large subunits contained in most of the CRISPR-Cas systems could be homologous to Cas10 proteins which contain a polymerase-like Palm domain and are predicted to be enzymatically active in Type III CRISPR-Cas systems but inactivated in Type I systems. These findings, the fact that the CRISPR polymerases, RAMPs and Cas2 all contain core RRM domains, and distinct gene arrangements in the three types of CRISPR-Cas systems together provide for a simple scenario for origin and evolution of the CRISPR-Cas machinery. Under this scenario, the CRISPR-Cas system originated in thermophilic Archaea and subsequently spread horizontally among prokaryotes. Conclusions Because of the extreme diversity of CRISPR-Cas systems, in-depth sequence and structure comparison continue to

  1. Substrate generation for endonucleases of CRISPR/cas systems.

    PubMed

    Zoephel, Judith; Dwarakanath, Srivatsa; Richter, Hagen; Plagens, André; Randau, Lennart

    2012-01-01

    The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) (1-3). Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems (4). The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster (5). The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs (6-8). These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence (9). A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity(10). Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA (11,12) . These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases (4). Here, we present methods to generate crRNAs and precursor-cRNAs for

  2. Costs of CRISPR-Cas-mediated resistance in Streptococcus thermophilus

    PubMed Central

    Vale, Pedro F.; Lafforgue, Guillaume; Gatchitch, Francois; Gardan, Rozenn; Moineau, Sylvain; Gandon, Sylvain

    2015-01-01

    CRISPR-Cas is a form of adaptive sequence-specific immunity in microbes. This system offers unique opportunities for the study of coevolution between bacteria and their viral pathogens, bacteriophages. A full understanding of the coevolutionary dynamics of CRISPR-Cas requires knowing the magnitude of the cost of resisting infection. Here, using the gram-positive bacterium Streptococcus thermophilus and its associated virulent phage 2972, a well-established model system harbouring at least two type II functional CRISPR-Cas systems, we obtained different fitness measures based on growth assays in isolation or in pairwise competition. We measured the fitness cost associated with different components of this adaptive immune system: the cost of Cas protein expression, the constitutive cost of increasing immune memory through additional spacers, and the conditional costs of immunity during phage exposure. We found that Cas protein expression is particularly costly, as Cas-deficient mutants achieved higher competitive abilities than the wild-type strain with functional Cas proteins. Increasing immune memory by acquiring up to four phage-derived spacers was not associated with fitness costs. In addition, the activation of the CRISPR-Cas system during phage exposure induces significant but small fitness costs. Together these results suggest that the costs of the CRISPR-Cas system arise mainly due to the maintenance of the defence system. We discuss the implications of these results for the evolution of CRISPR-Cas-mediated immunity. PMID:26224708

  3. Protein engineering of Cas9 for enhanced function

    PubMed Central

    Oakes, Benjamin L.; Nadler, Dana C.; Savage, David F.

    2015-01-01

    CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complimentary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of challenges regarding Cas9 specificity, efficiency, fusion protein function, and spatiotemporal control within the cell remain. In this work, we develop a platform for constructing novel proteins to address these open questions. We demonstrate methods to either screen or select active Cas9 mutants and use the screening technique to isolate functional Cas9 variants with a heterologous PDZ domain inserted directly into the protein. As a proof of concept, these methods lay the groundwork for the future construction of diverse Cas9 proteins. Straightforward and accessible techniques for genetic editing are helping to elucidate biology in new and exciting ways; a platform to engineer new functionalities into Cas9 will help forge the next generation of genome modifying tools. PMID:25398355

  4. CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri.

    PubMed

    Oh, Jee-Hwan; van Pijkeren, Jan-Peter

    2014-01-01

    Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR-Cas systems, such as the Streptococcus pyogenes CRISPR-Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR-Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR-Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR-Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR-Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR-Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR-Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria. PMID:25074379

  5. Expanding the catalog of cas genes with metagenomes.

    PubMed

    Zhang, Quan; Doak, Thomas G; Ye, Yuzhen

    2014-02-01

    The CRISPR (clusters of regularly interspaced short palindromic repeats)-Cas adaptive immune system is an important defense system in bacteria, providing targeted defense against invasions of foreign nucleic acids. CRISPR-Cas systems consist of CRISPR loci and cas (CRISPR-associated) genes: sequence segments of invaders are incorporated into host genomes at CRISPR loci to generate specificity, while adjacent cas genes encode proteins that mediate the defense process. We pursued an integrated approach to identifying putative cas genes from genomes and metagenomes, combining similarity searches with genomic neighborhood analysis. Application of our approach to bacterial genomes and human microbiome datasets allowed us to significantly expand the collection of cas genes: the sequence space of the Cas9 family, the key player in the recently engineered RNA-guided platforms for genome editing in eukaryotes, is expanded by at least two-fold with metagenomic datasets. We found genes in cas loci encoding other functions, for example, toxins and antitoxins, confirming the recently discovered potential of coupling between adaptive immunity and the dormancy/suicide systems. We further identified 24 novel Cas families; one novel family contains 20 proteins, all identified from the human microbiome datasets, illustrating the importance of metagenomics projects in expanding the diversity of cas genes. PMID:24319142

  6. Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice

    PubMed Central

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2016-01-01

    Recent reports of CRISPR- (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) mediated heritable mutagenesis in plants highlight the need for accuracy of the mutagenesis directed by this system. Off-target mutations are an important issue when considering functional gene analysis, as well as the molecular breeding of crop plants with large genome size, i.e. with many duplicated genes, and where the whole-genome sequence is still lacking. In mammals, off-target mutations can be suppressed by using Cas9 paired nickases together with paired guide RNAs (gRNAs). However, the performance of Cas9 paired nickases has not yet been fully assessed in plants. Here, we analyzed on- and off-target mutation frequency in rice calli and regenerated plants using Cas9 nuclease or Cas9 nickase with paired gRNAs. When Cas9 paired nickases were used, off-target mutations were fully suppressed in rice calli and regenerated plants. However, on-target mutation frequency also decreased compared with that induced by the Cas9 paired nucleases system. Since the gRNA sequence determines specific binding of Cas9 protein–gRNA ribonucleoproteins at the targeted sequence, the on-target mutation frequency of Cas9 paired nickases depends on the design of paired gRNAs. Our results suggest that a combination of gRNAs that can induce mutations at high efficiency with Cas9 nuclease should be used together with Cas9 nickase. Furthermore, we confirmed that a combination of gRNAs containing a one nucleotide (1 nt) mismatch toward the target sequence could not induce mutations when expressed with Cas9 nickase. Our results clearly show the effectiveness of Cas9 paired nickases in delivering on-target specific mutations. PMID:26936792

  7. Efficient Mitochondrial Genome Editing by CRISPR/Cas9

    PubMed Central

    Jo, Areum; Ham, Sangwoo; Lee, Gum Hwa; Lee, Yun-Il; Kim, SangSeong; Lee, Yun-Song; Shin, Joo-Ho; Lee, Yunjong

    2015-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing. PMID:26448933

  8. 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

    2016-07-01

    The CRISPR/Cas technology is enabling targeted genome editing in multiple organisms with unprecedented accuracy and specificity by using RNA-guided nucleases. A critical point when planning a CRISPR/Cas experiment is the design of the guide RNA (gRNA), which directs the nuclease and associated machinery to the desired genomic location. This gRNA has to fulfil the requirements of the nuclease and lack homology with other genome sites that could lead to off-target effects. Here we introduce the Breaking-Cas system for the design of gRNAs for CRISPR/Cas experiments, including those based in the Cas9 nuclease as well as others recently introduced. The server has unique features not available in other tools, including the possibility of using all eukaryotic genomes available in ENSEMBL (currently around 700), placing variable PAM sequences at 5' or 3' and setting the guide RNA length and the scores per nucleotides. It can be freely accessed at: http://bioinfogp.cnb.csic.es/tools/breakingcas, and the code is available upon request. PMID:27166368

  9. 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. PMID:26470680

  10. Inducible in vivo genome editing with CRISPR/Cas9

    PubMed Central

    O'Rourke, Kevin P; Muley, Ashlesha; Kastenhuber, Edward R; Livshits, Geulah; Tschaharganeh, Darjus F; Socci, Nicholas D; Lowe, Scott W

    2015-01-01

    CRISPR/Cas9-based genome editing enables the rapid genetic manipulation of any genomic locus without the need for gene targeting by homologous recombination. Here we describe a conditional transgenic approach that allows temporal control of CRISPR/Cas9 activity for inducible genome editing in adult mice. We show that doxycycline-regulated Cas9 induction enables widespread gene disruption in multiple tissues and that limiting the duration of Cas9 expression or using a Cas9D10A (Cas9n) variant, can regulate the frequency and size of target gene modifications, respectively. Further, we show that the inducible CRISPR (iCRISPR) system can be used effectively to create biallelic mutation in multiple target loci and thus, provides a flexible and fast platform to study loss of function phenotypes in vivo. PMID:25690852

  11. Intrication temporelle et communication quantique

    NASA Astrophysics Data System (ADS)

    Bussieres, Felix

    Quantum communication is the art of transferring a quantum state from one place to another and the study of tasks that can be accomplished with it. This thesis is devoted to the development of tools and tasks for quantum communication in a real-world setting. These were implemented using an underground optical fibre link deployed in an urban environment. The technological and theoretical innovations presented here broaden the range of applications of time-bin entanglement through new methods of manipulating time-bin qubits, a novel model for characterizing sources of photon pairs, new ways of testing non-locality and the design and the first implementation of a new loss-tolerant quantum coin-flipping protocol. Manipulating time-bin qubits. A single photon is an excellent vehicle in which a qubit, the fundamental unit of quantum information, can be encoded. In particular, the time-bin encoding of photonic qubits is well suited for optical fibre transmission. Before this thesis, the applications of quantum communication based on the time-bin encoding were limited due to the lack of methods to implement arbitrary operations and measurements. We have removed this restriction by proposing the first methods to realize arbitrary deterministic operations on time-bin qubits as well as single qubit measurements in an arbitrary basis. We applied these propositions to the specific case of optical measurement-based quantum computing and showed how to implement the feedforward operations, which are essential to this model. This therefore opens new possibilities for creating an optical quantum computer, but also for other quantum communication tasks. Characterizing sources of photon pairs. Experimental quantum communication requires the creation of single photons and entangled photons. These two ingredients can be obtained from a source of photon pairs based on non-linear spontaneous processes. Several tasks in quantum communication require a precise knowledge of the properties of the source being used. We developed and implemented a fast and simple method to characterize a source of photon pairs. This method is well suited for a realistic setting where experimental conditions, such as channel transmittance, may fluctuate, and for which the characterization of the source has to be done in real time. Testing the non-locality of time-bin entanglement. Entanglement is a resource needed for the realization of many important tasks in quantum communication. It also allows two physical systems to be correlated in a way that cannot be explained by classical physics; this manifestation of entanglement is called non-locality. We built a source of time-bin entangled photonic qubits and characterized it with the new methods implementing arbitrary single qubit measurements that we developed. This allowed us to reveal the non-local nature of our source of entanglement in ways that were never implemented before. It also opens the door to study previously untested features of non-locality using this source. Theses experiments were performed in a realistic setting where quantum (non-local) correlations were observed even after transmission of one of the entangled qubits over 12.4 km of an underground optical fibre. Flipping quantum coins. Quantum coin-flipping is a quantum cryptographic primitive proposed in 1984, that is when the very first steps of quantum communication were being taken, where two players alternate in sending classical and quantum information in order to generate a shared random bit. The use of quantum information is such that a potential cheater cannot force the outcome to his choice with certainty. Classically, however, one of the players can always deterministically choose the outcome. Unfortunately, the security of all previous quantum coin-flipping protocols is seriously compromised in the presence of losses on the transmission channel, thereby making this task impractical. We found a solution to this problem and obtained the first loss-tolerant quantum coin-flipping protocol whose security is independent of the amount of the losses. We have also experimentally demonstrated our loss-tolerant protocol using our source of time-bin entanglement combined with our arbitrary single qubit measurement methods. This experiment took place in a realistic setting where qubits travelled over an underground optical fibre link. This new task thus joins quantum key distribution as a practical application of quantum communication. Keywords. quantum communication, photonics, time-bin encoding, source of photon pairs, heralded single photon source, entanglement, non-locality, time-bin entanglement, hybrid entanglement, quantum network, quantum cryptography, quantum coin-flipping, measurement-based quantum computation, telecommunication, optical fibre, nonlinear optics.

  12. Evaporated CaS thin films for AC electroluminescence devices

    NASA Astrophysics Data System (ADS)

    Kobayashi, H.; Tanaka, S.; Shanker, V.; Shiiki, M.; Deguchi, H.

    1985-08-01

    The growth behavior of evaporated CaS thin films has been investigated to achieve bright electroluminescence. The crystallinity of CaS films is improved with substrate temperature and for temperatures higher than 300°C, the films orient to the (200) plane. Sulfur coevaporation further helps to form a more perfect film even at lower temperatures. A CaS: Ce,Cl electroluminescent thin film device has been fabricated with a brightness of 650 cd/m 2.

  13. Foreign DNA capture during CRISPR–Cas adaptive immunity

    PubMed Central

    Nuñez, James K.; Harrington, Lucas B.; Kranzusch, Philip J.; Engelman, Alan N.; Doudna, Jennifer A.

    2015-01-01

    Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30–40 base pair (bp) lengths into clustered regularly interspaced short palindromic repeats (CRISPR) loci as spacer segments1–6. The universally conserved Cas1–Cas2 integrase complex catalyzes spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases7–13. How the Cas1–Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1–Cas2 complex bound to cognate 33 nucleotide (nt) protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3′–OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo2–4. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1–Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci. PMID:26503043

  14. CRISPR-Cas9-guided Genome Engineering in C. elegans

    PubMed Central

    Kim, Hyun-Min; Colaiácovo, Monica P.

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is successfully being used for efficient and targeted genome editing in various organisms including the nematode C. elegans. Recent studies developed various CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: non-homologous end joining and homologous recombination. Here we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning and injection methods required for delivering Cas9, sgRNAs and repair template DNA into the C. elegans germline. PMID:27366893

  15. Foreign DNA capture during CRISPR-Cas adaptive immunity.

    PubMed

    Nuñez, James K; Harrington, Lucas B; Kranzusch, Philip J; Engelman, Alan N; Doudna, Jennifer A

    2015-11-26

    Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30-40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments. The universally conserved Cas1-Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1-Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1-Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3'-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci. PMID:26503043

  16. Optical Control of CRISPR/Cas9 Gene Editing

    PubMed Central

    Hemphill, James; Borchardt, Erin K.; Brown, Kalyn; Asokan, Aravind; Deiters, Alexander

    2016-01-01

    The CRISPR/Cas9 system has emerged as an important tool in biomedical research for a wide range of applications, with significant potential for genome engineering and gene therapy. In order to achieve conditional control of the CRISPR/Cas9 system, a genetically encoded light-activated Cas9 was engineered through the site-specific installation of a caged lysine amino acid. Several potential lysine residues were identified as viable caging sites that can be modified to optically control Cas9 function, as demonstrated through optical activation and deactivation of both exogenous and endogenous gene function. PMID:25905628

  17. Cas9 as a versatile tool for engineering biology

    PubMed Central

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

    2014-01-01

    RNA-guided Cas9 nucleases derived from clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have dramatically transformed our ability to edit the genomes of diverse organisms. We believe tools and techniques based on Cas9, a single unifying factor capable of colocalizing RNA, DNA and protein, will grant unprecedented control over cellular organization, regulation and behavior. Here we describe the Cas9 targeting methodology, detail current and prospective engineering advances and suggest potential applications ranging from basic science to the clinic. PMID:24076990

  18. Tenth anniversary of CAS ONLINE service : What CAS services should be in the new era of chemical information

    NASA Astrophysics Data System (ADS)

    Kostakos, Charles N.

    Chemical Abstracts Service celebrated 10th anniversary of CAS online information service in 1990. A speech given on the occasion reviewed history of the CAS ONLINE, in relation to its most important benefits for scientists and engineers. The development of STN international, the network through which CAS ONLINE is accessible around the world, was also discussed in the speech. The CAS ONLINE now contains a wide variety of files relating to chemical field including CA file, Registry file. CA previews,. CASREACT, CIN. MARPAT, etc for supplying chemical information worldwide.

  19. Superorbital variability of the X-ray flux in the Be-donor binaries SXP 138, GX-304, and γ Cas

    NASA Astrophysics Data System (ADS)

    Chashkina, A. A.; Abolmasov, P. K.; Biryukov, A. V.; Shakura, N. I.

    2015-06-01

    RXTE observations of the X-ray binary systems SXP 138, GX-304, and γ Cas in 1997-2011 have shown for the first time that these objects (X-ray binaries with Be donors) display X-ray flux variations on timescales of ˜1000 days. This timescale is about 10 times longer than their orbital periods, and is comparable to the total time of the observations. The observed variations are apparently not strictly periodic and represent stochastic variability, as is characteristic of such systems in the optical. γ Cas is considered as an example. The series of optical observations of this system available in the AAVSO database covers 78 years, and is much longer than the timescale of the variability studied. Our analysis of this series has shown that γ Cas variability on a timescale of tens of years is predominantly stochastic with a power-law spectrum.

  20. Cas9 Variants Expand the Target Repertoire in Caenorhabditis elegans.

    PubMed

    Bell, Ryan T; Fu, Becky X H; Fire, Andrew Z

    2016-02-01

    The proliferation of CRISPR/Cas9-based methods in Caenorhabditis elegans has enabled efficient genome editing and precise genomic tethering of Cas9 fusion proteins. Experimental designs using CRISPR/Cas9 are currently limited by the need for a protospacer adjacent motif (PAM) in the target with the sequence NGG. Here we report the characterization of two modified Cas9 proteins in C. elegans that recognize NGA and NGCG PAMs. We found that each variant could stimulate homologous recombination with a donor template at multiple loci and that PAM specificity was comparable to that of wild-type Cas9. To directly compare effectiveness, we used CRISPR/Cas9 genome editing to generate a set of assay strains with a common single-guide RNA (sgRNA) target sequence, but that differ in the juxtaposed PAM (NGG, NGA, or NGCG). In this controlled setting, we determined that the NGA PAM Cas9 variant can be as effective as wild-type Cas9. We similarly edited a genomic target to study the influence of the base following the NGA PAM. Using four strains with four NGAN PAMs differing only at the fourth position and adjacent to the same sgRNA target, we observed that efficient homologous replacement was attainable with any base in the fourth position, with an NGAG PAM being the most effective. In addition to demonstrating the utility of two Cas9 mutants in C. elegans and providing reagents that permit CRISPR/Cas9 experiments with fewer restrictions on potential targets, we established a means to benchmark the efficiency of different Cas9::PAM combinations that avoids variations owing to differences in the sgRNA sequence. PMID:26680661

  1. Recent Advances in Genome Editing Using CRISPR/Cas9

    PubMed Central

    Ding, Yuduan; Li, Hong; Chen, Ling-Ling; Xie, Kabin

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 technology, and discuss their implications for plant research. To date, targeted gene knockout using the Cas9/gRNA system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR/Cas9 system can also be used for sequence-specific mutagenesis/integration and transcriptional control of target genes. We also discuss off-target effects and the constraint that the protospacer-adjacent motif (PAM) puts on CRISPR/Cas9 genome engineering. To address these problems, a number of bioinformatic tools are available to help design specific gRNAs, and new Cas9 variants and orthologs with high fidelity and alternative PAM specificities have been engineered. Owing to these recent efforts, the CRISPR/Cas9 system is becoming a revolutionary and flexible tool for genome engineering. Adoption of the CRISPR/Cas9 technology in plant research would enable the investigation of plant biology at an unprecedented depth and create innovative applications in precise crop breeding. PMID:27252719

  2. Recent Progress in CRISPR/Cas9 Technology.

    PubMed

    Mei, Yue; Wang, Yan; Chen, Huiqian; Sun, Zhong Sheng; Ju, Xing-Da

    2016-02-20

    The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, a simple and efficient tool for genome editing, has experienced rapid progress in its technology and applicability in the past two years. Here, we review the recent advances in CRISPR/Cas9 technology and the ways that have been adopted to expand our capacity for precise genome manipulation. First, we introduce the mechanism of CRISPR/Cas9, including its biochemical and structural implications. Second, we highlight the latest improvements in the CRISPR/Cas9 system, especially Cas9 protein modifications for customization. Third, we review its current applications, in which the versatile CRISPR/Cas9 system was employed to edit the genome, epigenome, or RNA of various organisms. Although CRISPR/Cas9 allows convenient genome editing accompanied by many benefits, we should not ignore the significant ethical and biosafety concerns that it raises. Finally, we discuss the prospective applications and challenges of several promising techniques adapted from CRISPR/Cas9. PMID:26924689

  3. 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.

  4. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  5. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  6. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  7. Teaching Undergraduate Mathematics Using CAS Technology: Issues and Prospects

    ERIC Educational Resources Information Center

    Tobin, Patrick C.; Weiss, Vida

    2016-01-01

    The use of handheld CAS technology in undergraduate mathematics courses in Australia is paradoxically shrinking under sustained disapproval or disdain from the professional mathematics community. Mathematics education specialists argue with their mathematics colleagues over a range of issues in course development and this use of CAS or even…

  8. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  9. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  10. CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells.

    PubMed

    Ryan, Owen W; Poddar, Snigdha; Cate, Jamie H D

    2016-01-01

    This protocol describes a method for CRISPR-Cas9-mediated genome editing that results in scarless and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule that is used to repair the chromosomal DNA damage by homology-directed repair. For target specificity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA sequence within the sgRNA cassette. This CRISPR-Cas9 protocol includes methods for (1) cloning the unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides, and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial polyploid isolates. Therefore, this CRISPR-Cas9-based DNA integration protocol is achievable by virtually any yeast genetics and molecular biology laboratory. PMID:27250940

  11. Interacting Parallel Constructions of Knowledge in a CAS Context

    ERIC Educational Resources Information Center

    Kidron, Ivy; Dreyfus, Tommy

    2010-01-01

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

  12. Semiotic and Discursive Variables in CAS-Based Didactical Engineering.

    ERIC Educational Resources Information Center

    Winslow, Carl

    2003-01-01

    Presents a semiotic analysis of the potential of computer algebra systems (CAS) for enabling mathematical activity on a conceptual level higher than usual. Illustrates examples of theoretical points from a development project in the context of a first year university course in calculus. Discusses how CAS may be used in a didactical analysis and in…

  13. From Calculus to Dynamical Systems through DGS and CAS

    ERIC Educational Resources Information Center

    García, Jeanett López; Zamudio, Jorge Javier Jiménez

    2015-01-01

    Several factors have motivated the use of CAS or DGS in the teaching-learning process, such as: the development of new technologies, the availability of computers, and the widespread use of the Internet, among others. Even more, the trend to include CAS and DGS in the curricula of some undergraduate studies has resulted in the instruction of the…

  14. 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. PMID:27340770

  15. Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae.

    PubMed

    Generoso, Wesley Cardoso; Gottardi, Manuela; Oreb, Mislav; Boles, Eckhard

    2016-08-01

    CRISPR-Cas has become a powerful technique for genetic engineering of yeast. Here, we present an improved version by using only one single plasmid expressing Cas9 and one or two guide-RNAs. A high gene deletion efficiency was achieved even with simultaneous recombination cloning of the plasmid and deletion in industrial strains. PMID:27327211

  16. 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

  17. Conditional Control of CRISPR/Cas9 Function.

    PubMed

    Zhou, Wenyuan; Deiters, Alexander

    2016-04-25

    The recently discovered CRISPR/Cas9 endonuclease system, comprised of a guide RNA for the recognition of a DNA target and the Cas9 nuclease protein for binding and processing the target, has been extensively studied and has been widely applied in genome editing, synthetic biology, and transcriptional modulation in cells and animals. Toward more precise genomic modification and further expansion of the CRISPR/Cas9 system as a spatiotemporally controlled gene regulatory system, several approaches of conditional activation of Cas9 function using small molecules and light have recently been developed. These methods have led to improvements in the genome editing specificity of the CRISPR/Cas9 system and enabled its activation with temporal and spatial precision. PMID:26996256

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

    PubMed Central

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

    2016-01-01

    Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells. PMID:26870061

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

    PubMed

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

    2016-01-01

    Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells. PMID:26870061

  20. An Approach to the Study of Systems of Equations with Geogebra: Learning Opportunities Provided by the Integration of CAS View: Story of a Workshop Experience with Teachers

    ERIC Educational Resources Information Center

    Alejandra, Almirón; Fernando, Bifano; Leonardo, Lupinacci

    2015-01-01

    Solving systems of equations at school, at least in Argentina, is usually a task that students are given as a series of techniques that "allow" them to find a solution. How to overcome educational obstacles that are generated from a fragmented approach of knowledge? What can DGS do, in particular the CAS environment? What epistemic and…

  1. Implementing the CAS Standards: The Implementation of the CAS Standards in Student Affairs as a Comprehensive Assessment Approach

    ERIC Educational Resources Information Center

    Dorman, Jesse A.

    2012-01-01

    The increasing use of the CAS standards as a comprehensive assessment approach in divisions of student affairs necessitates a more in-depth understanding of how the CAS standards are being implemented in these settings. In response to increasing calls for improvement, accountability and professionalism in student affairs (Bresciani, 2006; Cooper…

  2. Interference activity of a minimal Type I CRISPR–Cas system from Shewanella putrefaciens

    PubMed Central

    Dwarakanath, Srivatsa; Brenzinger, Susanne; Gleditzsch, Daniel; Plagens, André; Klingl, Andreas; Thormann, Kai; Randau, Lennart

    2015-01-01

    Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs. PMID:26350210

  3. Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells

    PubMed Central

    Böttcher, Romy; Hollmann, Manuel; Merk, Karin; Nitschko, Volker; Obermaier, Christina; Philippou-Massier, Julia; Wieland, Isabella; Gaul, Ulrike; Förstemann, Klaus

    2014-01-01

    The ability to edit the genome is essential for many state-of-the-art experimental paradigms. Since DNA breaks stimulate repair, they can be exploited to target site-specific integration. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9 system from Streptococcus pyogenes has been harnessed into an efficient and programmable nuclease for eukaryotic cells. We thus combined DNA cleavage by cas9, the generation of homologous recombination donors by polymerase chain reaction (PCR) and transient depletion of the non-homologous end joining factor lig4. Using cultured Drosophila melanogaster S2-cells and the phosphoglycerate kinase gene as a model, we reached targeted integration frequencies of up to 50% in drug-selected cell populations. Homology arms as short as 29 nt appended to the PCR primer resulted in detectable integration, slightly longer extensions are beneficial. We confirmed established rules for S. pyogenes cas9 sgRNA design and demonstrate that the complementarity region allows length variation and 5′-extensions. This enables generation of U6-promoter fusion templates by overlap-extension PCR with a standardized protocol. We present a series of PCR template vectors for C-terminal protein tagging and clonal Drosophila S2 cell lines with stable expression of a myc-tagged cas9 protein. The system can be used for epitope tagging or reporter gene knock-ins in an experimental setup that can in principle be fully automated. PMID:24748663

  4. Pilomatricome: étude de 22 cas

    PubMed Central

    Nasreddine, Fatima Zahra; Hali, Fouzia; Chiheb, Soumiya

    2016-01-01

    Le pilomatricome est une tumeur cutanée fréquente et bénigne du follicule pileux chez l'enfant. C'est une tumeur annexielle souvent méconnue et confondue avec d'autres lésions cutanées. Les localisations habituelles sont la tête et le cou. Le but de ce travail est de rapporter une série de 22 cas comportant des formes inhabituelles colligées au service de dermatologie sur une période allant de Janvier 2006 jusqu'au Mai 2015. L’étude a concerné 16 femmes et 6 hommes. La moyenne d’âge était de 23,3 ans (4-80 ans). La localisation cervico faciale a été observée dans 12 cas, 2 patients avaient des localisations multiples, un garçon de 4ans avait une localisation au niveau fronto-temporal et une fillette de 14 ans avait une localisation au niveau du visage et de l'avant-bras, et un patient de 48 ans avait une localisation sous unguéale. L'aspect clinique était typique dans tous les cas avec des nodules sous cutanés de consistance pierreuse. Tous les patients ont bénéficié d'une exérèse des nodules sous anesthésie locale. L’étude histologique était en faveur d'un épithélioma momifié de Malherbe d'exérèse complète sans signes de malignité. Aucun patient n'a présenté de rechute. L'originalité de notre étude réside dans la présence de localisations exceptionnelles au niveau latéro-vertébral, des membres et sous-unguéale, l’âge de survenue inhabituel à 80 ans et la présence de localisations multiples signalées chez 2 enfants. PMID:27516819

  5. CRISPR/Cas9 in Genome Editing and Beyond.

    PubMed

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

    2016-06-01

    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. PMID:27145843

  6. 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. PMID:27072635

  7. 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

  8. HPCCP/CAS Workshop Proceedings 1998

    NASA Technical Reports Server (NTRS)

    Schulbach, Catherine; Mata, Ellen (Editor); Schulbach, Catherine (Editor)

    1999-01-01

    This publication is a collection of extended abstracts of presentations given at the HPCCP/CAS (High Performance Computing and Communications Program/Computational Aerosciences Project) Workshop held on August 24-26, 1998, at NASA Ames Research Center, Moffett Field, California. The objective of the Workshop was to bring together the aerospace high performance computing community, consisting of airframe and propulsion companies, independent software vendors, university researchers, and government scientists and engineers. The Workshop was sponsored by the HPCCP Office at NASA Ames Research Center. The Workshop consisted of over 40 presentations, including an overview of NASA's High Performance Computing and Communications Program and the Computational Aerosciences Project; ten sessions of papers representative of the high performance computing research conducted within the Program by the aerospace industry, academia, NASA, and other government laboratories; two panel sessions; and a special presentation by Mr. James Bailey.

  9. Adaptation in CRISPR-Cas Systems.

    PubMed

    Sternberg, Samuel H; Richter, Hagen; Charpentier, Emmanuelle; Qimron, Udi

    2016-03-17

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in prokaryotes. The system preserves memories of prior infections by integrating short segments of foreign DNA, termed spacers, into the CRISPR array in a process termed adaptation. During the past 3 years, significant progress has been made on the genetic requirements and molecular mechanisms of adaptation. Here we review these recent advances, with a focus on the experimental approaches that have been developed, the insights they generated, and a proposed mechanism for self- versus non-self-discrimination during the process of spacer selection. We further describe the regulation of adaptation and the protein players involved in this fascinating process that allows bacteria and archaea to harbor adaptive immunity. PMID:26949040

  10. Characterization and evolution of Salmonella CRISPR-Cas systems.

    PubMed

    Shariat, Nikki; Timme, Ruth E; Pettengill, James B; Barrangou, Rodolphe; Dudley, Edward G

    2015-02-01

    Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, although no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12% of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9%) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggest that Salmonella CRISPR-Cas systems are no longer immunogenic; rather, their impressive conservation indicates they may have an alternative function in Salmonella. PMID:25479838

  11. CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani

    PubMed Central

    Zhang, Wen-Wei

    2015-01-01

    ABSTRACT The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani. PMID:26199327

  12. 41 CFR 102-33.435 - What CAS cost and utilization data must we report?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 41 Public Contracts and Property Management 3 2012-01-01 2012-01-01 false What CAS cost and... Services (cas) Cost and Utilization Data § 102-33.435 What CAS cost and utilization data must we report? You must report the costs and flying hours for each CAS aircraft you hire. You must also report...

  13. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false If we hire CAS, what are... § 102-33.130 If we hire CAS, what are our management responsibilities? If you hire CAS, you are... agreements; (b) Accounting for the cost of your aircraft and services hired as CAS; (c) Accounting for use...

  14. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 41 Public Contracts and Property Management 3 2014-01-01 2014-01-01 false Who must report CAS cost... (cas) Cost and Utilization Data § 102-33.440 Who must report CAS cost and utilization data? Executive agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization...

  15. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 41 Public Contracts and Property Management 3 2011-01-01 2011-01-01 false If we hire CAS, what are... § 102-33.130 If we hire CAS, what are our management responsibilities? If you hire CAS, you are... agreements; (b) Accounting for the cost of your aircraft and services hired as CAS; (c) Accounting for use...

  16. 41 CFR 102-33.435 - What CAS cost and utilization data must we report?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 41 Public Contracts and Property Management 3 2014-01-01 2014-01-01 false What CAS cost and... Services (cas) Cost and Utilization Data § 102-33.435 What CAS cost and utilization data must we report? You must report the costs and flying hours for each CAS aircraft you hire. You must also report...

  17. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 41 Public Contracts and Property Management 3 2013-07-01 2013-07-01 false Who must report CAS cost... (cas) Cost and Utilization Data § 102-33.440 Who must report CAS cost and utilization data? Executive agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization...

  18. 41 CFR 102-33.435 - What CAS cost and utilization data must we report?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false What CAS cost and... Services (cas) Cost and Utilization Data § 102-33.435 What CAS cost and utilization data must we report? You must report the costs and flying hours for each CAS aircraft you hire. You must also report...

  19. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 41 Public Contracts and Property Management 3 2013-07-01 2013-07-01 false If we hire CAS, what are... § 102-33.130 If we hire CAS, what are our management responsibilities? If you hire CAS, you are... agreements; (b) Accounting for the cost of your aircraft and services hired as CAS; (c) Accounting for use...

  20. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false Who must report CAS cost... (cas) Cost and Utilization Data § 102-33.440 Who must report CAS cost and utilization data? Executive agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization...

  1. 41 CFR 102-33.435 - What CAS cost and utilization data must we report?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 41 Public Contracts and Property Management 3 2013-07-01 2013-07-01 false What CAS cost and... Services (cas) Cost and Utilization Data § 102-33.435 What CAS cost and utilization data must we report? You must report the costs and flying hours for each CAS aircraft you hire. You must also report...

  2. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 41 Public Contracts and Property Management 3 2011-01-01 2011-01-01 false Who must report CAS cost... (cas) Cost and Utilization Data § 102-33.440 Who must report CAS cost and utilization data? Executive agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization...

  3. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 41 Public Contracts and Property Management 3 2012-01-01 2012-01-01 false If we hire CAS, what are... § 102-33.130 If we hire CAS, what are our management responsibilities? If you hire CAS, you are... agreements; (b) Accounting for the cost of your aircraft and services hired as CAS; (c) Accounting for use...

  4. 41 CFR 102-33.435 - What CAS cost and utilization data must we report?

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 41 Public Contracts and Property Management 3 2011-01-01 2011-01-01 false What CAS cost and... Services (cas) Cost and Utilization Data § 102-33.435 What CAS cost and utilization data must we report? You must report the costs and flying hours for each CAS aircraft you hire. You must also report...

  5. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 41 Public Contracts and Property Management 3 2012-01-01 2012-01-01 false Who must report CAS cost... (cas) Cost and Utilization Data § 102-33.440 Who must report CAS cost and utilization data? Executive agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization...

  6. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 41 Public Contracts and Property Management 3 2014-01-01 2014-01-01 false If we hire CAS, what are... § 102-33.130 If we hire CAS, what are our management responsibilities? If you hire CAS, you are... agreements; (b) Accounting for the cost of your aircraft and services hired as CAS; (c) Accounting for use...

  7. Grossesse intra murale à propos d'un cas

    PubMed Central

    de Tové, Kofi-Mensa Savi; Salifou, Kabibou; Imorou, Rachidi Sidi; Biaou, Olivier; Boco, Vicentia

    2015-01-01

    La grossesse intra-murale est la variété la plus rare de grossesse extra-utérine. Il s'agit de la localisation de l’œuf dans l’épaisseur du myomètre. En cas de retard diagnostic, l’évolution peut être catastrophique avec rupture utérine et hémorragie cataclysmique. L’échographie permet dans certains cas un diagnostic pré opératoire. Les auteurs rapportent un cas survenu chez une patiente aux antécédents de curetage. PMID:26448812

  8. Introducing the TI-Nspire CAS Learning Technology

    NASA Astrophysics Data System (ADS)

    Osbourne, Michael

    2008-03-01

    Engage your students! In this hands-on workshop, we will demonstrate how TI's newest graphing technology can be integrated into the physics classroom. Come experience both the TI-NspireT CAS Handheld and the TI- NspireT CAS Computer Software. The new TI-Nspire learning technology has been developed to allow students to explore physics and to better understand concepts through the manipulation of complex formulas,graphing, spreadsheets, data analysis, and simulations. During the workshop you will receive an overview of the technology, sample activities, along with the opportunity to experience how TI-Nspire CAS can be effectively integrated into the physics classroom. Limited to 20 participants.

  9. Determination of the Physical Dimensions of μ Cas

    NASA Astrophysics Data System (ADS)

    Bach, K.; Kang, W.

    2015-07-01

    We investigate the physical properties of the nearby astrometric binary μ Cas based on a spectroscopic analysis and evolutionary calculations. The chemical composition of the μ Cas system has been determined based on high resolution spectroscopy from BOES. Combining our spectroscopic analysis with observations from Hipparcos and CHARA, we determine the evolutionary status of μ Cas. With well-constrained stellar parameters, the internal structure and frequency modes have also been calculated. The ultimate goal of this study is to describe the physical processes inside stars through a comprehensive modeling.

  10. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity.

    PubMed

    Barrangou, Rodolphe; Marraffini, Luciano A

    2014-04-24

    Clustered regularly interspaced short palindromic repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing and can be repurposed for numerous DNA targeting applications including transcriptional control. PMID:24766887

  11. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity

    PubMed Central

    Barrangou, Rodolphe; Marraffini, Luciano A.

    2014-01-01

    Summary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing, and can be repurposed for numerous DNA targeting applications including transcriptional control. PMID:24766887

  12. Engineering the Caenorhabditis elegans genome with CRISPR/Cas9.

    PubMed

    Waaijers, Selma; Boxem, Mike

    2014-08-01

    The development in early 2013 of CRISPR/Cas9-based genome engineering promises to dramatically advance our ability to alter the genomes of model systems at will. A single, easily produced targeting RNA guides the Cas9 endonuclease to a specific DNA sequence where it creates a double strand break. Imprecise repair of the break can yield mutations, while homologous recombination with a repair template can be used to effect specific changes to the genome. The tremendous potential of this system led several groups to independently adapt it for use in Caenorhabditiselegans, where it was successfully used to generate mutations and to create tailored genome changes through homologous recombination. Here, we review the different approaches taken to adapt CRISPR/Cas9 for C. elegans, and provide practical guidelines for CRISPR/Cas9-based genome engineering. PMID:24685391

  13. The Structural Biology of CRISPR-Cas Systems

    PubMed Central

    Jiang, Fuguo; Doudna, Jennifer A.

    2015-01-01

    Prokaryotic CRISPR-Cas genomic loci encode RNA-mediated adaptive immune systems that bear some functional similarities with eukaryotic RNA interference. Acquired and heritable immunity against bacteriophage and plasmids begins with integration of ~30 base pair foreign DNA sequences into the host genome. CRISPR-derived transcripts assemble with CRISPR-associated (Cas) proteins to target complementary nucleic acids for degradation. Here we review recent advances in the structural biology of these targeting complexes, with a focus on structural studies of the multisubunit Type I CRISPR RNA-guided surveillance and the Cas9 DNA endonuclease found in Type II CRISPR-Cas systems. These complexes have distinct structures that are each capable of site-specific double-stranded DNA binding and local helix unwinding. PMID:25723899

  14. CAS Standards and Guidelines for Student Services/Development Programs.

    ERIC Educational Resources Information Center

    NACADA Journal, 1986

    1986-01-01

    The Council for the Advancement of Standards for Student Services/Development Programs (CAS) developed and adopted standards and interpretive guidelines for specific functional areas of student services/development programs within postsecondary educational institutions. These guidelines are presented. (MLW)

  15. CRISPR-Cas9-Guided Genome Engineering in C. elegans.

    PubMed

    Kim, Hyun-Min; Colaiácovo, Monica P

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is successfully being used for efficient and targeted genome editing in various organisms, including the nematode C. elegans. Recent studies have developed various CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: non-homologous end joining and homologous recombination. Here we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning, as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the C. elegans germline. © 2016 by John Wiley & Sons, Inc. PMID:27366893

  16. Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity.

    PubMed

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

    2016-08-01

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

  17. An updated evolutionary classification of CRISPR-Cas systems.

    PubMed

    Makarova, Kira S; Wolf, Yuri I; Alkhnbashi, Omer S; Costa, Fabrizio; Shah, Shiraz A; Saunders, Sita J; Barrangou, Rodolphe; Brouns, Stan J J; Charpentier, Emmanuelle; Haft, Daniel H; Horvath, Philippe; Moineau, Sylvain; Mojica, Francisco J M; Terns, Rebecca M; Terns, Michael P; White, Malcolm F; Yakunin, Alexander F; Garrett, Roger A; van der Oost, John; Backofen, Rolf; Koonin, Eugene V

    2015-11-01

    The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized. PMID:26411297

  18. Assiniboine Series.

    ERIC Educational Resources Information Center

    Allen, Minerva

    This series of illustrated booklets presents 13 Indian stories in a bilingual format of English and Assiniboine, an Indian tribal language. Written on the first grade level, the stories have the following titles: (1) "Orange Tree in Lodgepole"; (2) "Pretty Flower"; (3) Inktomi and the Rock"; (4) "Inktomi and the Ducks"; (5) "Inktomi and the…

  19. Target specificity of the CRISPR-Cas9 system

    PubMed Central

    Wu, Xuebing; Kriz, Andrea J.; Sharp, Phillip A.

    2015-01-01

    The CRISPR-Cas9 system, naturally a defense mechanism in prokaryotes, has been repurposed as an RNA-guided DNA targeting platform. It has been widely used for genome editing and transcriptome modulation, and has shown great promise in correcting mutations in human genetic diseases. Off-target effects are a critical issue for all of these applications. Here we review the current status on the target specificity of the CRISPR-Cas9 system. PMID:25722925

  20. Comparison of Cas9 activators in multiple species.

    PubMed

    Chavez, Alejandro; Tuttle, Marcelle; Pruitt, Benjamin W; Ewen-Campen, Ben; Chari, Raj; Ter-Ovanesyan, Dmitry; Haque, Sabina J; Cecchi, Ryan J; Kowal, Emma J K; Buchthal, Joanna; Housden, Benjamin E; Perrimon, Norbert; Collins, James J; Church, George

    2016-07-01

    Several programmable transcription factors exist based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems, and we assess the role of cooperativity in maximizing gene expression. PMID:27214048

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

    PubMed

    Peng, Rongxue; Lin, Guigao; Li, Jinming

    2016-04-01

    Recently, a novel technique named the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)9 system has been rapidly developed. This genome editing tool has improved our ability tremendously with respect to exploring the pathogenesis of diseases and correcting disease mutations, as well as phenotypes. With a short guide RNA, Cas9 can be precisely directed to target sites, and functions as an endonuclease to efficiently produce breaks in DNA double strands. Over the past 30 years, CRISPR has evolved from the 'curious sequences of unknown biological function' into a promising genome editing tool. As a result of the incessant development in the CRISPR/Cas9 system, Cas9 co-expressed with custom guide RNAs has been successfully used in a variety of cells and organisms. This genome editing technology can also be applied to synthetic biology, functional genomic screening, transcriptional modulation and gene therapy. However, although CRISPR/Cas9 has a broad range of action in science, there are several aspects that affect its efficiency and specificity, including Cas9 activity, target site selection and short guide RNA design, delivery methods, off-target effects and the incidence of homology-directed repair. In the present review, we highlight the factors that affect the utilization of CRISPR/Cas9, as well as possible strategies for handling any problems. Addressing these issues will allow us to take better advantage of this technique. In addition, we also review the history and rapid development of the CRISPR/Cas system from the time of its initial discovery in 2012. PMID:26535798

  2. CRISPR-Cas9 Based Engineering of Actinomycetal Genomes.

    PubMed

    Tong, Yaojun; Charusanti, Pep; Zhang, Lixin; Weber, Tilmann; Lee, Sang Yup

    2015-09-18

    Bacteria of the order Actinomycetales are one of the most important sources of pharmacologically active and industrially relevant secondary metabolites. Unfortunately, many of them are still recalcitrant to genetic manipulation, which is a bottleneck for systematic metabolic engineering. To facilitate the genetic manipulation of actinomycetes, we developed a highly efficient CRISPR-Cas9 system to delete gene(s) or gene cluster(s), implement precise gene replacements, and reversibly control gene expression in actinomycetes. We demonstrate our system by targeting two genes, actIORF1 (SCO5087) and actVB (SCO5092), from the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3(2). Our CRISPR-Cas9 system successfully inactivated the targeted genes. When no templates for homology-directed repair (HDR) were present, the site-specific DNA double-strand breaks (DSBs) introduced by Cas9 were repaired through the error-prone nonhomologous end joining (NHEJ) pathway, resulting in a library of deletions with variable sizes around the targeted sequence. If templates for HDR were provided at the same time, precise deletions of the targeted gene were observed with near 100% frequency. Moreover, we developed a system to efficiently and reversibly control expression of target genes, deemed CRISPRi, based on a catalytically dead variant of Cas9 (dCas9). The CRISPR-Cas9 based system described here comprises a powerful and broadly applicable set of tools to manipulate actinomycetal genomes. PMID:25806970

  3. Rational design of a split-Cas9 enzyme complex

    PubMed Central

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

    2015-01-01

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications. PMID:25713377

  4. Internal guide RNA interactions interfere with Cas9-mediated cleavage.

    PubMed

    Thyme, Summer B; Akhmetova, Laila; Montague, Tessa G; Valen, Eivind; Schier, Alexander F

    2016-01-01

    The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9-gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9-gRNA complex formation. PMID:27282953

  5. Rational design of a split-Cas9 enzyme complex

    DOE PAGESBeta

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

    2015-02-23

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

  6. Internal guide RNA interactions interfere with Cas9-mediated cleavage

    PubMed Central

    Thyme, Summer B.; Akhmetova, Laila; Montague, Tessa G.; Valen, Eivind; Schier, Alexander F.

    2016-01-01

    The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9–gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9–gRNA complex formation. PMID:27282953

  7. Rational design of a split-Cas9 enzyme complex

    SciTech Connect

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

    2015-02-23

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

  8. Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9

    PubMed Central

    Kaya, Hidetaka; Mikami, Masafumi; Endo, Akira; Endo, Masaki; Toki, Seiichi

    2016-01-01

    The CRISPR/Cas9 system is an efficient and convenient tool for genome editing in plants. Cas9 nuclease derived from Streptococcus pyogenes (Sp) is commonly used in this system. Recently, Staphylococcus aureus Cas9 (SaCas9)-mediated genome editing was reported in human cells and Arabidopsis. Because SaCas9 (1053 a.a.) is smaller than SpCas9 (1368 a.a.), SaCas9 could have substantial advantages for delivering and expressing Cas9 protein, especially using virus vectors. Since the protospacer adjacent motif (PAM) sequence of SaCas9 (5′-NNGRRT-3′) differs from that of SpCas9 (5′-NGG-3′), the use of this alternative Cas9 nuclease could expand the selectivity at potential cleavage target sites of the CRISPR/Cas9 system. Here we show that SaCas9 can mutagenize target sequences in tobacco and rice with efficiencies similar to those of SpCas9. We also analyzed the base preference for ‘T’ at the 6th position of the SaCas9 PAM. Targeted mutagenesis efficiencies in target sequences with non-canonical PAMs (5′-NNGRRV-3′) were much lower than those with a canonical PAM (5′-NNGRRT-3′). The length of target sequence recognized by SaCas9 is one or two nucleotides longer than that recognized by SpCas9. Taken together, our results demonstrate that SaCas9 has higher sequence recognition capacity than SpCas9 and is useful for reducing off-target mutations in crop. PMID:27226350

  9. Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9.

    PubMed

    Kaya, Hidetaka; Mikami, Masafumi; Endo, Akira; Endo, Masaki; Toki, Seiichi

    2016-01-01

    The CRISPR/Cas9 system is an efficient and convenient tool for genome editing in plants. Cas9 nuclease derived from Streptococcus pyogenes (Sp) is commonly used in this system. Recently, Staphylococcus aureus Cas9 (SaCas9)-mediated genome editing was reported in human cells and Arabidopsis. Because SaCas9 (1053 a.a.) is smaller than SpCas9 (1368 a.a.), SaCas9 could have substantial advantages for delivering and expressing Cas9 protein, especially using virus vectors. Since the protospacer adjacent motif (PAM) sequence of SaCas9 (5'-NNGRRT-3') differs from that of SpCas9 (5'-NGG-3'), the use of this alternative Cas9 nuclease could expand the selectivity at potential cleavage target sites of the CRISPR/Cas9 system. Here we show that SaCas9 can mutagenize target sequences in tobacco and rice with efficiencies similar to those of SpCas9. We also analyzed the base preference for 'T' at the 6th position of the SaCas9 PAM. Targeted mutagenesis efficiencies in target sequences with non-canonical PAMs (5'-NNGRRV-3') were much lower than those with a canonical PAM (5'-NNGRRT-3'). The length of target sequence recognized by SaCas9 is one or two nucleotides longer than that recognized by SpCas9. Taken together, our results demonstrate that SaCas9 has higher sequence recognition capacity than SpCas9 and is useful for reducing off-target mutations in crop. PMID:27226350

  10. A non-inheritable maternal Cas9-based multiple-gene editing system in mice

    PubMed Central

    Sakurai, Takayuki; Kamiyoshi, Akiko; Kawate, Hisaka; Mori, Chie; Watanabe, Satoshi; Tanaka, Megumu; Uetake, Ryuichi; Sato, Masahiro; Shindo, Takayuki

    2016-01-01

    The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9 overexpression (Cas9 mice). The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome. The efficiency of such maCas9-based genome editing was comparable to that of zygote microinjection–based genome editing widely used at present. Furthermore, we demonstrated a novel approach to create “Cas9 transgene-free” gene-modified mice using non-Tg (+/+) zygotes carrying maCas9. The maCas9 protein in mouse zygotes edited nine target loci simultaneously after injection with nine different gRNAs alone. Cas9 mouse-derived zygotes have the potential to facilitate the creation of genetically modified animals carrying the Cas9 transgene, enabling repeatable genome engineering and the production of Cas9 transgene-free mice. PMID:26817415

  11. A non-inheritable maternal Cas9-based multiple-gene editing system in mice.

    PubMed

    Sakurai, Takayuki; Kamiyoshi, Akiko; Kawate, Hisaka; Mori, Chie; Watanabe, Satoshi; Tanaka, Megumu; Uetake, Ryuichi; Sato, Masahiro; Shindo, Takayuki

    2016-01-01

    The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9 overexpression (Cas9 mice). The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome. The efficiency of such maCas9-based genome editing was comparable to that of zygote microinjection-based genome editing widely used at present. Furthermore, we demonstrated a novel approach to create "Cas9 transgene-free" gene-modified mice using non-Tg (+/+) zygotes carrying maCas9. The maCas9 protein in mouse zygotes edited nine target loci simultaneously after injection with nine different gRNAs alone. Cas9 mouse-derived zygotes have the potential to facilitate the creation of genetically modified animals carrying the Cas9 transgene, enabling repeatable genome engineering and the production of Cas9 transgene-free mice. PMID:26817415

  12. A Class Exercise: Studying the Eclipsing Binary Star RZ Cas Through Visual Observations

    NASA Astrophysics Data System (ADS)

    Balonek, T. J.; Davis, S. M.

    2000-05-01

    As part of the sophomore-junior level "Astronomical Techniques" course at Colgate University, students learn just how much science they can do with simple tools: a pair of binoculars, a clock, and pencil and paper. The students study the Algol type visual eclipsing binary star system RZ Cassiopeiae: observing and making a light curve for the primary minimum, determining the time of minimum using several techniques, calculating the binary star system's orbital period, and determining changes in the system's period over a thirty year interval by constructing an O-C curve. Through a series of preparatory exercises, the students learn how to read star maps and use the unaided eye, binoculars and telescopes to locate star fields and make visual magnitude measurements. By making multiple measurements of stars in the field of RZ Cas on several nights, the students determine the accuracy they can achieve in estimating the visual magnitude of a star -- typically 0.2 magnitude. (Some students even accidentally discover that one of the stars in the field is a variable star!) With this experience, the students use binoculars to observe the four hour primary eclipse of RZ Cas (magnitude 6.2 - 7.7), making magnitude measurements every five minutes. A light curve is then plotted. Several methods are used to determine the time of minimum, which is then converted to heliocentric Julian day. Using times of minima determined by former students (and the instructor) in previous years dating from 1968 to the present, the students determine the average period to a tenth of a second second. By constructing an O-C curve from the class's data and that obtained by the AAVSO, changes in the period of RZ Cas are noticeable -- possibly due to mass transfer in the system. It will be interesting for future classes to build on this knowledge using the primitive tools of our not so distant past.

  13. Biograph™ series.

    PubMed

    2005-01-01

    Medical imaging is one of the fastest growing areas in healthcare. Combined imaging systems are at the forefront of this growth, transforming the industry by uniting functional imaging such as PET, with diagnostic multi-slice CT, thus providing an anatomical map for accurate localization, diagnosis and, finally, treatment of diseases. Dedication and consistent innovation enables Siemens Medical Solutions to offer the Biograph™ PET/CT series. This imaging system offers a high technological standard in line with excellent image quality and speed for maximum patient comfort and increased diagnostic confidence for physicians. PMID:16395982

  14. Tuning CAS Application using AIMS: An Automated Instrumentation and Monitoring System

    NASA Technical Reports Server (NTRS)

    Mehra, P.; Sarukkai, S.; Schmidt, M.; Schulbach, C.; VanVoorst, B.; Yan, Jerry; Woodrow, Thomas (Technical Monitor)

    1994-01-01

    To bring together NASA's scientists and engineers and their counterparts in industry, other government agencies, and academia working in the Computational AeroSciences (CAS) field. This workshop is part of the technology transfer plan of the High Performance Computing and Communications Program (HPCCP). Specific objectives of this Workshop are to: (1) communicate the goals and objectives of HPCCP in the area of CAS; (2) promote and disseminate CAS technology within the appropriate technical communities, including NASA, industry, academia, and other government labs; (3) help promote synergy among CAS scientists; and (4) permit feedback from peer researchers in issues pacing the CAS field in general and the HPCCP CAS program in particular.

  15. Spectroscopic studies of three Cepheids with high positive pulsation period increments: SZ Cas, BY Cas, and RU Sct

    NASA Astrophysics Data System (ADS)

    Usenko, I. A.; Klochkova, V. G.

    2015-07-01

    Three high-resolution spectra have been taken at different times with the 6-m SAO RAS telescope (LYNX and PFES spectrographs) for three Cepheids exhibiting high positive period increments: the small-amplitude (DCEPS) SZ Cas and BY Cas and the classical (DCEP) RU Sct. SZ Cas and RU Sct are members of the Galactic open clusters χ and h Per and Trump 35, respectively. Analysis of the spectra has shown that the interstellar Na I D1 and D2 lines in all objects are considerably stronger than the atmospheric ones and are redshifted in SZ Cas and BY Cas and blushifted in RU Sct. The core of the H α absorption line in BY Cas has an asymmetric knifelike shape, while RU Sct exhibits an intense emission in the blue wing of this line. Such phenomena are observed in long-period Cepheids and bright hypergiants with an extended envelope. In this case, the strong Mg Ib 5183.62 Å and Ba II 5853.67, 6141.713, and 6496.90 Å lines with low χlow in SZ Cas and RU Sct also show characteristic knifelike profiles with an asymmetry in the red region, while the Ba II 4934.095 Å line shows similar profiles in the blue one. The absorption lines of neutral atoms and singly ionized metals with different lowerlevel excitation potentials exhibit different degrees of asymmetry: from a pronounced one with secondary components in BY Cas (similar to those in the small-amplitude Cepheid BG Cru pulsating in the first overtone and having an envelope) to its insignificance or virtual absence in SZ Cas and RU Sct. Analysis of the secular changes in mean T eff determined from photometric color indices and spectra over the last 55 years for these stars has revealed periodic fluctuations of 200 K for SZ Cas and BY Cas and 500 K for RU Sct. For SZ Cas and RU Sct, T eff determined in some years from some color indices show much lower values, which together with the temperature fluctuations can be associated with mass loss and dust formation. Based on these facts, we hypothesize the existence of

  16. Pyo-pneumothorax tuberculeux: à propos de 18 cas

    PubMed Central

    Hicham, Souhi; Hanane, El Ouazzani; Hicham, Janah; Ismaïl, Rhorfi; Ahmed, Abid

    2016-01-01

    Le pyo-pneumothorax tuberculeux est une complication rare mais grave de la tuberculose pulmonaire évolutive. Nous rapportons une série de 18 cas de pyo-pneumothorax tuberculeux colligés au service de Pneumo-Phtisiologie de l'Hôpital Militaire d'Instruction Mohammed V de Rabat entre janvier 2005 et décembre 2009. Il s'agit de 15 hommes et 3 femmes d’âge moyen de 35 ans ±7 ans. 4 patients étaient diabétiques. Le tabagisme était retrouvé chez 9 cas. Le pyo-pneumothorax était du coté droit dans 13 cas. La radiographie thoracique avait montré des lésions cavitaires chez 15 patients et des lésions étendues et bilatérales chez 8 cas. La recherche de BK dans le liquide de tubage gastrique était positive chez 16 cas. Un drainage thoracique associé à un traitement antituberculeux selon le régime 2SRHZ/7RH et une kinésithérapie respiratoire ont été instaurés chez tous les cas. La durée moyenne de drainage pleural était de 4 semaines. Chez 3 cas on avait noté la persistance de la suppuration pleurale ayant nécessité une toilette pleurale sous thoracoscopie avec pleurectomie et exérèse pulmonaire limitée emportant la lésion parenchymateuse tuberculeuse et la persistance d'une volumineuse poche pleurale avec trouble ventilatoire restrictif ayant nécessité une décortication pleurale chirurgicale chez deux cas. L’évolution était favorable avec pachypleurite séquellaire minime chez le reste des cas. Le pyo-pneumothorax tuberculeux est une forme grave, qui est souvent en rapport avec une tuberculose cavitaire active. L’évolution est généralement trainante malgré le traitement antituberculeux et le drainage thoracique, d'où la nécessité d'un diagnostic et un traitement précoce de toute forme de tuberculose. PMID:27583090

  17. Repurposing CRISPR/Cas9 for in situ functional assays

    PubMed Central

    Malina, Abba; Mills, John R.; Cencic, Regina; Yan, Yifei; Fraser, James; Schippers, Laura M.; Paquet, Marilène; Dostie, Josée; Pelletier, Jerry

    2013-01-01

    RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel “all-in-one” lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an “all-in-one” system to track disrupted Trp53 in chemoresistant lymphomas in the Eμ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens. PMID:24298059

  18. SD-CAS: Spin Dynamics by Computer Algebra System.

    PubMed

    Filip, Xenia; Filip, Claudiu

    2010-11-01

    A computer algebra tool for describing the Liouville-space quantum evolution of nuclear 1/2-spins is introduced and implemented within a computational framework named Spin Dynamics by Computer Algebra System (SD-CAS). A distinctive feature compared with numerical and previous computer algebra approaches to solving spin dynamics problems results from the fact that no matrix representation for spin operators is used in SD-CAS, which determines a full symbolic character to the performed computations. Spin correlations are stored in SD-CAS as four-entry nested lists of which size increases linearly with the number of spins into the system and are easily mapped into analytical expressions in terms of spin operator products. For the so defined SD-CAS spin correlations a set of specialized functions and procedures is introduced that are essential for implementing basic spin algebra operations, such as the spin operator products, commutators, and scalar products. They provide results in an abstract algebraic form: specific procedures to quantitatively evaluate such symbolic expressions with respect to the involved spin interaction parameters and experimental conditions are also discussed. Although the main focus in the present work is on laying the foundation for spin dynamics symbolic computation in NMR based on a non-matrix formalism, practical aspects are also considered throughout the theoretical development process. In particular, specific SD-CAS routines have been implemented using the YACAS computer algebra package (http://yacas.sourceforge.net), and their functionality was demonstrated on a few illustrative examples. PMID:20843716

  19. Measurement of Flux Density of Cas A at Low Frequencies

    NASA Astrophysics Data System (ADS)

    Patil, Ajinkya; Fisher, R.

    2012-01-01

    Cas A is used as a flux calibrator throughout the radio spectrum. Therefore it is important to know the spectral and secular variations in its flux density. Earlier observations by Scott et. al. (1969) and Baars et. al. (1972) suggested a secular decrease in flux density of Cas A at a rate of about 1% per year at all frequencies. However later observations by Erickson & Perley (1975) and Read (1977) indicated anomalously high flux from Cas A at 38 MHz. Also, these observations suggested that the original idea of faster decay of the flux density rate at low frequencies may be in error or that something more complex than simple decay is affecting the flux density at low frequencies. The source changes at 38 MHz still remains a mystery. We intend to present the results of follow up observations made from 1995 to 1998 with a three element interferometer in Green Bank operating in frequency range 30 to 120 MHz. We will discuss the problems at such low frequencies due to large beamwidth and unstable ionosphere. We will also discuss the strategies we have used so far to to find the flux density of Cas A by calculating the ratio of flux density of Cas A to that of Cyg A, assuming flux density of Cyg A to be constant. Above mentioned work was performed in summer student program sponsored by National Radio Astronomy Observatory.

  20. RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses.

    PubMed

    Baker, Oliver; Gupta, Ashish; Obst, Mandy; Zhang, Youming; Anastassiadis, Konstantinos; Fu, Jun; Stewart, A Francis

    2016-01-01

    A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated recombineering pipeline for fluent generation of long arm targeting constructs is also presented. PMID:27216209

  1. RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses

    PubMed Central

    Baker, Oliver; Gupta, Ashish; Obst, Mandy; Zhang, Youming; Anastassiadis, Konstantinos; Fu, Jun; Stewart, A. Francis

    2016-01-01

    A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated recombineering pipeline for fluent generation of long arm targeting constructs is also presented. PMID:27216209

  2. Displacement of p130Cas from focal adhesions links actomyosin contraction to cell migration.

    PubMed

    Machiyama, Hiroaki; Hirata, Hiroaki; Loh, Xia Kun; Kanchi, Madhu Mathi; Fujita, Hideaki; Tan, Song Hui; Kawauchi, Keiko; Sawada, Yasuhiro

    2014-08-15

    Cell adhesion complexes provide platforms where cell-generated forces are transmitted to the extracellular matrix (ECM). Tyrosine phosphorylation of focal adhesion proteins is crucial for cells to communicate with the extracellular environment. However, the mechanisms that transmit actin cytoskeletal motion to the extracellular environment to drive cell migration are poorly understood. We find that the movement of p130Cas (Cas, also known as BCAR1), a mechanosensor at focal adhesions, correlates with actin retrograde flow and depends upon actomyosin contraction and phosphorylation of the Cas substrate domain (CasSD). This indicates that CasSD phosphorylation underpins the physical link between Cas and the actin cytoskeleton. Fluorescence recovery after photobleaching (FRAP) experiments reveal that CasSD phosphorylation, as opposed to the association of Cas with Src, facilitates Cas displacement from adhesion complexes in migrating cells. Furthermore, the stabilization of Src-Cas binding and inhibition of myosin II, both of which sustain CasSD phosphorylation but mitigate Cas displacement from adhesion sites, retard cell migration. These results indicate that Cas promotes cell migration by linking actomyosin contractions to the adhesion complexes through a dynamic interaction with Src as well as through the phosphorylation-dependent association with the actin cytoskeleton. PMID:24928898

  3. DNA fragment editing of genomes by CRISPR/Cas9.

    PubMed

    Jinhuan, Li; Jia, Shou; Qiang, Wu

    2015-10-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system from bacteria and archaea emerged recently as a new powerful technology of genome editing in virtually any organism. Due to its simplicity and cost effectiveness, a revolutionary change of genetics has occurred. Here, we summarize the recent development of DNA fragment editing methods by CRISPR/Cas9 and describe targeted DNA fragment deletions, inversions, duplications, insertions, and translocations. The efficient method of DNA fragment editing provides a powerful tool for studying gene function, regulatory elements, tissue development, and disease progression. Finally, we discuss the prospects of CRISPR/Cas9 system and the potential applications of other types of CRISPR system. PMID:26496751

  4. CRISPR-Cas9: A Revolutionary Tool for Cancer Modelling.

    PubMed

    Torres-Ruiz, Raul; Rodriguez-Perales, Sandra

    2015-01-01

    The cancer-modelling field is now experiencing a conversion with the recent emergence of the RNA-programmable CRISPR-Cas9 system, a flexible methodology to produce essentially any desired modification in the genome. Cancer is a multistep process that involves many genetic mutations and other genome rearrangements. Despite their importance, it is difficult to recapitulate the degree of genetic complexity found in patient tumors. The CRISPR-Cas9 system for genome editing has been proven as a robust technology that makes it possible to generate cellular and animal models that recapitulate those cooperative alterations rapidly and at low cost. In this review, we will discuss the innovative applications of the CRISPR-Cas9 system to generate new models, providing a new way to interrogate the development and progression of cancers. PMID:26389881

  5. CRISPR-Cas9: A Revolutionary Tool for Cancer Modelling

    PubMed Central

    Torres-Ruiz, Raul; Rodriguez-Perales, Sandra

    2015-01-01

    The cancer-modelling field is now experiencing a conversion with the recent emergence of the RNA-programmable CRISPR-Cas9 system, a flexible methodology to produce essentially any desired modification in the genome. Cancer is a multistep process that involves many genetic mutations and other genome rearrangements. Despite their importance, it is difficult to recapitulate the degree of genetic complexity found in patient tumors. The CRISPR-Cas9 system for genome editing has been proven as a robust technology that makes it possible to generate cellular and animal models that recapitulate those cooperative alterations rapidly and at low cost. In this review, we will discuss the innovative applications of the CRISPR-Cas9 system to generate new models, providing a new way to interrogate the development and progression of cancers. PMID:26389881

  6. GENOME EDITING IN HUMAN CELLS USING CRISPR/CAS NUCLEASES

    PubMed Central

    Wyvekens, Nicolas; Tsai, Shengdar; Joung, J. Keith

    2016-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been broadly adopted for highly efficient genome editing in a variety of model organisms and human cell types. Unlike previous genome editing technologies such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas technology does not require complex protein engineering and can be utilized by any researcher proficient in basic molecular biology and cell culture techniques. Here we describe protocols for design and cloning of vectors expressing single or multiplex gRNAs, for transient transfection of human cell lines, and for quantitation of mutation frequencies by T7 Endonuclease I assay. These protocols also include guidance for using two improvements that increase the specificity of CRISPR/Cas nucleases: truncated gRNAs and dimeric RNA-guided FokI nucleases. PMID:26423589

  7. Applications of CRISPR-Cas systems in neuroscience

    PubMed Central

    Heidenreich, Matthias; Zhang, Feng

    2016-01-01

    Genome editing tools, and in particular those based on CRISPR-Cas systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in virtually any organism and cell type. These tools have opened the door to the development of new model systems for studying the complexity of the nervous system, including animal and stem cell-derived in vitro models. Precise and efficient gene editing using CRISPR-Cas systems has the potential to advance both basic and translational neuroscience research. PMID:26656253

  8. Mouse Genome Editing using CRISPR/Cas System

    PubMed Central

    Harms, Donald W; Quadros, Rolen M; Seruggia, Davide; Ohtsuka, Masato; Takahashi, Gou

    2015-01-01

    The availability of techniques to create desired genetic mutations has enabled the laboratory mouse as an extensively used model organism in biomedical research including human genetics. A new addition to this existing technical repertoire is the CRISPR/Cas system. Specifically, this system allows editing of the mouse genome much faster than the previously used techniques and more importantly multiple mutations can be created in a single experiment. Here we provide protocols for preparation of CRISPR/Cas reagents and microinjection into one cell mouse embryos to create knockout or knock-in mouse models. PMID:25271839

  9. Les sarcomes des tissus mous: à propos de 33 cas

    PubMed Central

    Abdou, Jiddou; Elkabous, Mustapha; M'rabti, Hind; Errihani, Hassan

    2015-01-01

    L'objectif de cette étude est de rapporter les particularités épidémiologiques, cliniques, histologiques, thérapeutiques et évolutives des sarcomes des tissus mous à l'Institut National d'Oncologie et de définir les facteurs influençant la survie des patients. C'est une étude rétrospective de 33 cas de sarcome des tissus mous, colligés entre janvier 2008 et décembre 2010. Les critères d’éligibilité étaient un âge supérieur à 16 ans, une épreuve histologique d'un sarcome des tissus mous à l'exclusion des tumeurs stromales gastro-intestinales (GIST). Les items recueillis étaient: épidémiologiques, cliniques, histologiques, Radiologiques, et thérapeutiques. Des analyses univariées puis multivariées ont été réalisées à la recherche de facteurs influençant la survie à 2 ans. Il s'agit de 33 cas, 17 Hommes et 16 Femmes, l’âge moyen était de 43,21 ans (Extrêmes= 18-76 ans). La tumeur était localisée aux extrémités dans 24 cas (72,72%). Le type histologique prédominant était le Liposarcome dans 9 cas (27,27%). Le stade tumoral était localisé dans 25 cas (75,8%), métastatique dans 8 cas (24,2%). Vingt-cinq tumeurs ont été traitées chirurgicalement dont 21 cas (84%) de chirurgie conservatrice et 4 cas (16%) de chirurgie radicale. La radiothérapie a été réalisée chez 10 patients (30,3%). La chimiothérapie a été faite chez 20 patients. En analyse univariée les facteurs pronostiques étaient l’âge (p=0,03) et le stade tumoral (p=0,09). L’âge et le stade tumoral sont des facteurs pronostiques influençant la survie des sarcomes des tissus mous. PMID:27022434

  10. High-throughput functional genomics using CRISPR-Cas9

    PubMed Central

    Shalem, Ophir; Sanjana, Neville E.; Zhang, Feng

    2015-01-01

    Forward genetic screens are powerful tools for the discovery and functional annotation of genetic elements. Recently, the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has been combined with genome-scale guide RNA libraries for unbiased, phenotypic screening. In this Review, we describe recent advances using Cas9 for genome-scale screens, including knockout approaches that inactivate genomic loci and strategies that modulate transcriptional activity. We discuss practical aspects of screen design, provide comparisons with RNA interference (RNAi) screening, and outline future applications and challenges. PMID:25854182

  11. Cas9 gRNA engineering for genome editing, activation and repression

    PubMed Central

    Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; Hall, Richard N; Chari, Raj; Ter-Ovanesyan, Dmitry; Qian, Jason; Pruitt, Benjamin W; Beal, Jacob; Vora, Suhani; Buchthal, Joanna; Kowal, Emma J K; Ebrahimkhani, Mohammad R; Collins, James J; Weiss, Ron; Church, George

    2015-01-01

    We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein. PMID:26344044

  12. Cas9 gRNA engineering for genome editing, activation and repression.

    PubMed

    Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; Hall, Richard N; Chari, Raj; Ter-Ovanesyan, Dmitry; Qian, Jason; Pruitt, Benjamin W; Beal, Jacob; Vora, Suhani; Buchthal, Joanna; Kowal, Emma J K; Ebrahimkhani, Mohammad R; Collins, James J; Weiss, Ron; Church, George

    2015-11-01

    We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein. PMID:26344044

  13. V845 Cas - ein RRab-Stern mit Blazhko-Effekt

    NASA Astrophysics Data System (ADS)

    Maintz, Gisela

    2015-02-01

    CCD observations of V845 Cas (RA= 23 26 15.02, DE= +57 23 55.5 (2000)) were obtained at my private observatory. For V845 Cas 13 maxima were gained, showing a light curve, that varies from epoch to epoch. The irregularity of the lightcurves of V845 Cas is due to the Blazhko effect. Revised elements are given as: V845 Cas Max = 2456221.3450 + 0.570845 * E.

  14. Functional annotation of native enhancers with a Cas9-histone demethylase fusion.

    PubMed

    Kearns, Nicola A; Pham, Hannah; Tabak, Barbara; Genga, Ryan M; Silverstein, Noah J; Garber, Manuel; Maehr, René

    2015-05-01

    Understanding of mammalian enhancers is limited by the lack of a technology to rapidly and thoroughly test the cell type-specific function. Here, we use a nuclease-deficient Cas9 (dCas9)-histone demethylase fusion to functionally characterize previously described and new enhancer elements for their roles in the embryonic stem cell state. Further, we distinguish the mechanism of action of dCas9-LSD1 at enhancers from previous dCas9-effectors. PMID:25775043

  15. The Cas6e ribonuclease is not required for interference and adaptation by the E. coli type I-E CRISPR-Cas system

    PubMed Central

    Semenova, Ekaterina; Kuznedelov, Konstantin; Datsenko, Kirill A.; Boudry, Pierre M.; Savitskaya, Ekaterina E.; Medvedeva, Sofia; Beloglazova, Natalia; Logacheva, Maria; Yakunin, Alexander F.; Severinov, Konstantin

    2015-01-01

    CRISPR-Cas are small RNA-based adaptive prokaryotic immunity systems protecting cells from foreign DNA or RNA. Type I CRISPR-Cas systems are composed of a multiprotein complex (Cascade) that, when bound to CRISPR RNA (crRNA), can recognize double-stranded DNA targets and recruit the Cas3 nuclease to destroy target-containing DNA. In the Escherichia coli type I-E CRISPR-Cas system, crRNAs are generated upon transcription of CRISPR arrays consisting of multiple palindromic repeats and intervening spacers through the function of Cas6e endoribonuclease, which cleaves at specific positions of repeat sequences of the CRISPR array transcript. Cas6e is also a component of Cascade. Here, we show that when mature unit-sized crRNAs are provided in a Cas6e-independent manner by transcription termination, the CRISPR-Cas system can function without Cas6e. The results should allow facile interrogation of various targets by type I-E CRISPR-Cas system in E. coli using unit-sized crRNAs generated by transcription. PMID:26013814

  16. CRISPR/Cas-Mediated Site-Specific Mutagenesis in Arabidopsis thaliana Using Cas9 Nucleases and Paired Nickases.

    PubMed

    Schiml, Simon; Fauser, Friedrich; Puchta, Holger

    2016-01-01

    The CRISPR/Cas system has recently become the most important tool for genome engineering due to its simple architecture that allows for rapidly changing the target sequence and its applicability to organisms throughout all kingdoms of life. The need for an easy-to-use and reliable nuclease is especially high in plant research, as precise genome modifications are almost impossible to achieve by Agrobacterium-mediated transformation and the regeneration of plants from protoplast cultures is very labor intensive. Here, we describe the application of the Cas9 nuclease to Arabidopsis thaliana for the induction of heritable targeted mutations, which may also be used for other plant species. To cover the concern for off-target activity, we also describe the generation of stable mutants using paired Cas9 nickases. PMID:27557689

  17. Creating Genome Modifications in C. elegans Using the CRISPR/Cas9 System.

    PubMed

    Calarco, John A; Friedland, Ari E

    2015-01-01

    The clustered, regularly interspaced, short, palindromic repeat (CRISPR)-associated (CAS) nuclease Cas9 has been used in many organisms to generate specific mutations and transgene insertions. Here we describe a method using the S. pyogenes Cas9 in C. elegans that provides a convenient and effective approach for making heritable changes to the worm genome. PMID:26423968

  18. 48 CFR 9904.412-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CAS Pension Harmonization Rule. 9904.412-64.1 Section 9904.412-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. Contractors or subcontractors that become subject... through 7 Notes (Note 1) Actuarial Accrued Liability $2,470,500 $14,225,000 2 CAS Actuarial Value...

  19. 48 CFR 9904.412-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CAS Pension Harmonization Rule. 9904.412-64.1 Section 9904.412-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. Contractors or subcontractors that become subject... through 7 Notes (Note 1) Actuarial Accrued Liability $2,470,500 $14,225,000 2 CAS Actuarial Value...

  20. 48 CFR 9904.412-60.1 - Illustrations-CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 7 2013-10-01 2012-10-01 true Illustrations-CAS Pension... AND COST ACCOUNTING STANDARDS COST ACCOUNTING STANDARDS 9904.412-60.1 Illustrations—CAS Pension... cost on or after the Applicability Date of the CAS Harmonization Rule. The illustrations present...

  1. 48 CFR 9904.412-60.1 - Illustrations-CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 7 2014-10-01 2014-10-01 false Illustrations-CAS Pension... AND COST ACCOUNTING STANDARDS COST ACCOUNTING STANDARDS 9904.412-60.1 Illustrations—CAS Pension... cost on or after the Applicability Date of the CAS Harmonization Rule. The illustrations present...

  2. 48 CFR 9904.412-60.1 - Illustrations-CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 7 2012-10-01 2012-10-01 false Illustrations-CAS Pension... AND COST ACCOUNTING STANDARDS COST ACCOUNTING STANDARDS 9904.412-60.1 Illustrations—CAS Pension... cost on or after the Applicability Date of the CAS Harmonization Rule. The illustrations present...

  3. 48 CFR 9904.413-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CAS Pension Harmonization Rule. 9904.413-64.1 Section 9904.413-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. The transition method for the CAS Pension Harmonization Rule under this Standard shall be in accordance with 9904.412.64.1 Transition Method for...

  4. Mismatch Negativity Responses in Children with a Diagnosis of Childhood Apraxia of Speech (CAS)

    ERIC Educational Resources Information Center

    Froud, Karen; Khamis-Dakwar, Reem

    2012-01-01

    Purpose: To evaluate whether a hypothesis suggesting that apraxia of speech results from phonological overspecification could be relevant for childhood apraxia of speech (CAS). Method: High-density EEG was recorded from 5 children with CAS and 5 matched controls, ages 5-8 years, with and without CAS, as they listened to randomized sequences of CV…

  5. 48 CFR 9904.413-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CAS Pension Harmonization Rule. 9904.413-64.1 Section 9904.413-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. The transition method for the CAS Pension Harmonization Rule under this Standard shall be in accordance with 9904.412.64.1 Transition Method for...

  6. 48 CFR 9904.413-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CAS Pension Harmonization Rule. 9904.413-64.1 Section 9904.413-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. The transition method for the CAS Pension Harmonization Rule under this Standard shall be in accordance with 9904.412.64.1 Transition Method for...

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

    ERIC Educational Resources Information Center

    Driver, David

    2012-01-01

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

  8. 48 CFR 9904.412-64.1 - Transition Method for the CAS Pension Harmonization Rule.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CAS Pension Harmonization Rule. 9904.412-64.1 Section 9904.412-64.1 Federal Acquisition Regulations... Transition Method for the CAS Pension Harmonization Rule. Contractors or subcontractors that become subject... through 7 Notes (Note 1) Actuarial Accrued Liability $2,470,500 $14,225,000 2 CAS Actuarial Value...

  9. On the Integration of Computer Algebra Systems (CAS) by Canadian Mathematicians: Results of a National Survey

    ERIC Educational Resources Information Center

    Buteau, Chantal; Jarvis, Daniel H.; Lavicza, Zsolt

    2014-01-01

    In this article, we outline the findings of a Canadian survey study (N = 302) that focused on the extent of computer algebra systems (CAS)-based technology use in postsecondary mathematics instruction. Results suggest that a considerable number of Canadian mathematicians use CAS in research and teaching. CAS use in research was found to be the…

  10. After 16 Years of Publishing Standards, Do CAS Standards Make a Difference?

    ERIC Educational Resources Information Center

    Arminio, Jan; Gochenauer, Patty

    2004-01-01

    Using members of professional associations who are a part of the Council for the Advancement of Standards in Higher Education (CAS) consortia as a sample, this study investigated who uses CAS Standards, how and why they are used, and whether CAS Standards are associated with enhanced student learning. Using a quantitative analysis, this study…

  11. The role of Cas8 in type I CRISPR interference

    PubMed Central

    Cass, Simon D.B.; Haas, Karina A.; Stoll, Britta; Alkhnbashi, Omer S.; Sharma, Kundan; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita; Bolt, Edward L.

    2015-01-01

    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. PMID:26182359

  12. 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

    2015-01-01

    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. PMID:26182359

  13. Guide RNAs: A Glimpse at the Sequences that Drive CRISPR-Cas Systems.

    PubMed

    Briner, Alexandra E; Barrangou, Rodolphe

    2016-01-01

    CRISPR-Cas systems provide adaptive immunity in bacteria and archaea. Although there are two main classes of CRISPR-Cas systems defined by gene content, interfering RNA biogenesis, and effector proteins, Type II systems have recently been exploited on a broad scale to develop next-generation genetic engineering and genome-editing tools. Conveniently, Type II systems are streamlined and rely on a single protein, Cas9, and a guide RNA molecule, comprised of a CRISPR RNA (crRNA) and trans-acting CRISPR RNA (tracrRNA), to achieve effective and programmable nucleic acid targeting and cleavage. Currently, most commercially available Cas9-based genome-editing tools use the CRISPR-Cas system from Streptococcus pyogenes (SpyCas9), although many orthogonal Type II systems are available for diverse and multiplexable genome engineering applications. Here, we discuss the biological significance of Type II CRISPR-Cas elements, including the tracrRNA, crRNA, Cas9, and protospacer-adjacent motif (PAM), and look at the native function of these elements to understand how they can be engineered, enhanced, and optimized for genome editing applications. Additionally, we discuss the basis for orthogonal Cas9 and guide RNA systems that would allow researchers to concurrently use multiple Cas9-based systems for different purposes. Understanding the native function of endogenous Type II CRISPR-Cas systems can lead to new Cas9 tool development to expand the genetic manipulation toolbox. PMID:27371605

  14. Apport de l'imagerie dans le diagnostic des sacroiliites infectieuses : à propos de 19 cas

    PubMed Central

    Abid, Hanen; Chaabouni, Salim; Frikha, Faten; Toumi, Nozha; Souissi, Basma; Lahiani, Dorra; Bahloul, Zouhir; Ben Mahfoudh, Khaireddine

    2014-01-01

    Les sacro-iliites infectieuses méritent d’être mieux connues. Leur diagnostic est souvent retardé en raison d'une symptomatologie trompeuse et des diffcultés d'exploration de l'articulation sacro-iliaque. Notre travail est basé sur une étude rétrospective portant sur les cas de SII, recueillis sur une période comprise entre 1997 et 2008 dans notre centre universitaire Sfax-Tunisie. Le diagnostic de sacro-iliite était retenu en présence d'arguments cliniques et radiologiques d'atteinte sacroiliaque. Nous rapportons dix neuf cas de sacroiliites infectieuses (10 hommes et 9 femmes), avec un âge moyen de 32 ans. L'atteinte était unilatérale dans tous les cas. Les radiographies standard faites dans tous les cas ont été suggestives dans 14 cas et normales dans les autres cas. La TDM faite dans 13 cas a montré, un abcès des parties molles dans 8 cas et un séquestre osseux dans 2 cas. L'IRM réalisée dans 8 cas, a objectivé une infiltration des parties molles dans tous les cas et un abcès dans 3 cas. Le germe a été identifié dans 9 cas (3 cas de tuberculose, 3 cas de brucellose, 2 sacro-iliites à pyogène et un cas de candidose). Cette identification était faite par biopsie dans 3 cas, hémocultures dans 2 cas, prélèvement au niveau de la porte d'entrée dans 1 cas et sérodiagnostic dans 3 cas. Pour les autres cas, l'origine pyogène a été retenue sur des arguments cliniques et biologiques. L'imagerie joue un rôle primordial dans le diagnostic précoce et l'orientation étiologique d'une sacroiliite infectieuse. PMID:25120884

  15. Problem Solving in Calculus with Symbolic Geometry and CAS

    ERIC Educational Resources Information Center

    Todd, Philip; Wiechmann, James

    2008-01-01

    Computer algebra systems (CAS) have been around for a number of years, as has dynamic geometry. Symbolic geometry software is new. It bears a superficial similarity to dynamic geometry software, but differs in that problems may be set up involving symbolic variables and constants, and measurements are given as symbolic expressions. Mathematical…

  16. CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans.

    PubMed

    Waaijers, Selma; Portegijs, Vincent; Kerver, Jana; Lemmens, Bennie B L G; Tijsterman, Marcel; van den Heuvel, Sander; Boxem, Mike

    2013-11-01

    The generation of genetic mutants in Caenorhabditis elegans has long relied on the selection of mutations in large-scale screens. Directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. Here, we adapt the CRISPR/Cas9 system to generate mutations at specific sites in the C. elegans genome. PMID:23979586

  17. CRISPR/Cas9 advances engineering of microbial cell factories.

    PubMed

    Jakočiūnas, Tadas; Jensen, Michael K; Keasling, Jay D

    2016-03-01

    One of the key drivers for successful metabolic engineering in microbes is the efficacy by which genomes can be edited. As such there are many methods to choose from when aiming to modify genomes, especially those of model organisms like yeast and bacteria. In recent years, clustered regularly interspaced palindromic repeats (CRISPR) and its associated proteins (Cas) have become the method of choice for precision genome engineering in many organisms due to their orthogonality, versatility and efficacy. Here we review the strategies adopted for implementation of RNA-guided CRISPR/Cas9 genome editing with special emphasis on their application for metabolic engineering of yeast and bacteria. Also, examples of how nuclease-deficient Cas9 has been applied for RNA-guided transcriptional regulation of target genes will be reviewed, as well as tools available for computer-aided design of guide-RNAs will be highlighted. Finally, this review will provide a perspective on the immediate challenges and opportunities foreseen by the use of CRISPR/Cas9 genome engineering and regulation in the context of metabolic engineering. PMID:26707540

  18. CRISPR-Cas9 genome editing in Drosophila

    PubMed Central

    Gratz, Scott J.; Rubinstein, C. Dustin; Harrison, Melissa M.; Wildonger, Jill; O’Connor-Giles, Kate M.

    2015-01-01

    The CRISPR-Cas9 system has transformed genome engineering of model organisms from possible to practical. CRISPR-Cas9 can be readily programmed to generate sequence-specific double-strand breaks that disrupt targeted loci when repaired by error-prone non-homologous end joining or to catalyze precise genome modification through homology-directed repair (HDR). Here we describe a streamlined approach for rapid and highly efficient engineering of the Drosophila genome via CRISPR-Cas9-mediated HDR. In this approach, transgenic flies expressing Cas9 are injected with plasmids to express guide RNAs (gRNAs) and positively marked donor templates. We detail target site selection; gRNA plasmid generation; donor template design and construction; and the generation, identification and molecular confirmation of engineered lines. We also present alternative approaches and highlight key considerations for experimental design. The approach outlined here can be used to rapidly and reliably generate a variety of engineered modifications, including genomic deletions and replacements, precise sequence edits, and incorporation of protein tags. PMID:26131852

  19. Indispensable Manual Calculation Skills in a CAS Environment.

    ERIC Educational Resources Information Center

    Herget, Wilfried; Heugl, Helmut; Kutzler, Bernhard; Lehmann, Eberhard

    Which manual calculation skills are still needed when students use graphic/symbolic calculators or computers with computer algebra systems (CAS)? What should students be able to do manually, i.e. just using paper and pencil? This text is the outcome of a two-day discussion on these questions, held by the four authors. Our answers and proposals are…

  20. Equivalent Expressions Using CAS and Paper-and-Pencil Techniques

    ERIC Educational Resources Information Center

    Fonger, Nicole L.

    2014-01-01

    How can the key concept of equivalent expressions be addressed so that students strengthen their representational fluency with symbols, graphs, and numbers? How can research inform the synergistic use of both paper-and-pencil analysis and computer algebra systems (CAS) in a classroom learning environment? These and other related questions have…

  1. Preparing Students to Take SOA/CAS Exam FM/2

    ERIC Educational Resources Information Center

    Marchand, Richard J.

    2014-01-01

    This paper provides suggestions for preparing students to take the actuarial examination on financial mathematics, SOA/CAS Exam FM/2. It is based on current practices employed at Slippery Rock University, a small public liberal arts university. Detailed descriptions of our Theory of Interest course and subsequent Exam FM/2 prep course are provided…

  2. Research Needed on the Use of CAS Standards and Guidelines.

    ERIC Educational Resources Information Center

    Creamer, Don G.

    2003-01-01

    This article suggests research projects that would extend the knowledge base about the use of Council for the Advancement of Standards in Higher Education (CAS) standards and guidelines in useful ways. Included are five research questions and specific research methodologies to guide researchers. (Contains 20 references.) (Author)

  3. Prospective Mathematics Teachers' Interactions with CAS-Based Textbook Elements

    ERIC Educational Resources Information Center

    Davis, Jon D.

    2015-01-01

    This study investigated how a group of 10 prospective secondary mathematics teachers (PST) read, evaluated, and adapted a textbook lesson involving the symbolic manipulation capabilities of computer algebra systems (CASS). PST read the entire lesson and tended to focus on the organizing question at the beginning of the student lesson and the CAS-S…

  4. Does CAS Use Disadvantage Girls in VCE Mathematics?

    ERIC Educational Resources Information Center

    Forgasz, Helen; Tan, Hazel

    2010-01-01

    In 2009, four mathematics subjects were offered at the year 12 level in the Victorian Certificate of Education (VCE). The two subjects at the intermediate level--Mathematical Methods and Mathematical Methods CAS--run in parallel, that is, a student can be enrolled in only one or the other, the choice being made at the school level. The curricular…

  5. Targeted mutagenesis in chicken using CRISPR/Cas9 system.

    PubMed

    Oishi, Isao; Yoshii, Kyoko; Miyahara, Daichi; Kagami, Hiroshi; Tagami, Takahiro

    2016-01-01

    The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens. Two egg white genes, ovalbumin and ovomucoid, were efficiently (>90%) mutagenized in cultured chicken primordial germ cells (PGCs) by transfection of circular plasmids encoding Cas9, a single guide RNA, and a gene encoding drug resistance, followed by transient antibiotic selection. We transplanted CRISPR-induced mutant-ovomucoid PGCs into recipient chicken embryos and established three germline chimeric roosters (G0). All of the roosters had donor-derived mutant-ovomucoid spermatozoa, and the two with a high transmission rate of donor-derived gametes produced heterozygous mutant ovomucoid chickens as about half of their donor-derived offspring in the next generation (G1). Furthermore, we generated ovomucoid homozygous mutant offspring (G2) by crossing the G1 mutant chickens. Taken together, these results demonstrate that the CRISPR/Cas9 system is a simple and effective gene-targeting method in chickens. PMID:27050479

  6. Targeted mutagenesis in chicken using CRISPR/Cas9 system

    PubMed Central

    Oishi, Isao; Yoshii, Kyoko; Miyahara, Daichi; Kagami, Hiroshi; Tagami, Takahiro

    2016-01-01

    The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens. Two egg white genes, ovalbumin and ovomucoid, were efficiently (>90%) mutagenized in cultured chicken primordial germ cells (PGCs) by transfection of circular plasmids encoding Cas9, a single guide RNA, and a gene encoding drug resistance, followed by transient antibiotic selection. We transplanted CRISPR-induced mutant-ovomucoid PGCs into recipient chicken embryos and established three germline chimeric roosters (G0). All of the roosters had donor-derived mutant-ovomucoid spermatozoa, and the two with a high transmission rate of donor-derived gametes produced heterozygous mutant ovomucoid chickens as about half of their donor-derived offspring in the next generation (G1). Furthermore, we generated ovomucoid homozygous mutant offspring (G2) by crossing the G1 mutant chickens. Taken together, these results demonstrate that the CRISPR/Cas9 system is a simple and effective gene-targeting method in chickens. PMID:27050479

  7. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

    PubMed

    Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian

    2016-06-01

    CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo. PMID:27130213

  8. Some Reflections on CAS Assisted Proofs of Theorems

    ERIC Educational Resources Information Center

    Dana-Picard, Thierry

    2005-01-01

    A mathematician's work consists of proving theorems, calculating, and making mathematics understandable. An assistant for all three components is a Computer Algebra System. We describe and discuss various CAS-assisted processes for proving theorems, and discuss the constraints which can appear regarding efficiency, confidence in the result and…

  9. Technological Discourse on CAS-Based Operative Knowledge

    ERIC Educational Resources Information Center

    Mann, Giora; Dana-Picard, Thierry; Zehavi, Nurit

    2007-01-01

    This article begins with a comparison of two groups of teachers, working on the same tasks in Analytic Geometry. One group has only basic experience in CAS-assisted problem solving, and the other group has extensive experience. The comparison is discussed in terms of the interplay between reflection, operative knowledge and execution. The findings…

  10. Duchenne muscular dystrophy: CRISPR/Cas9 treatment.

    PubMed

    Mendell, Jerry R; Rodino-Klapac, Louise R

    2016-05-01

    A novel approach to gene correction by genome editing shows great promise as a treatment for Duchenne muscular dystrophy (DMD). CRISPR/Cas9 delivered by adeno-associated virus to a mouse model for DMD demonstrated improvement in function and histology. PMID:26926391

  11. New Insight in Mathematics by Live CAS Documents.

    ERIC Educational Resources Information Center

    Cnop, Ivan

    Traditional education in mathematics is mostly a matter of hindsight, and many mathematics texts offer little opportunity for students and learners to gain insight. This paper shows how experiments in CAS (computer algebra systems) can lead to new ways of handling problems, new conjectures, new visualizations, new proofs, new correspondences…

  12. 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. PMID:27210042

  13. Production of genome-edited pluripotent stem cells and mice by CRISPR/Cas [Review].

    PubMed

    Horii, Takuro; Hatada, Izuho

    2016-03-31

    Clustered regularly at interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases, so-called CRISPR/Cas, was recently developed as an epoch-making genome engineering technology. This system only requires Cas9 nuclease and single-guide RNA complementary to a target locus. CRISPR/Cas enables the generation of knockout cells and animals in a single step. This system can also be used to generate multiple mutations and knockin in a single step, which is not possible using other methods. In this review, we provide an overview of genome editing by CRISPR/Cas in pluripotent stem cells and mice. PMID:26743444

  14. Mobile CRISPR/Cas-Mediated Bacteriophage Resistance in Lactococcus lactis

    PubMed Central

    Millen, Anne M.; Horvath, Philippe; Boyaval, Patrick; Romero, Dennis A.

    2012-01-01

    Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes. PMID:23240053

  15. Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses.

    PubMed

    Wang, Dan; Mou, Haiwei; Li, Shaoyong; Li, Yingxiang; Hough, Soren; Tran, Karen; Li, Jia; Yin, Hao; Anderson, Daniel G; Sontheimer, Erik J; Weng, Zhiping; Gao, Guangping; Xue, Wen

    2015-07-01

    CRISPR/Cas9 derived from the bacterial adaptive immunity pathway is a powerful tool for genome editing, but the safety profiles of in vivo delivered Cas9 (including host immune responses to the bacterial Cas9 protein) have not been comprehensively investigated in model organisms. Nonalcoholic steatohepatitis (NASH) is a prevalent human liver disease characterized by excessive fat accumulation in the liver. In this study, we used adenovirus (Ad) vector to deliver a Streptococcus pyogenes-derived Cas9 system (SpCas9) targeting Pten, a gene involved in NASH and a negative regulator of the PI3K-AKT pathway, in mouse liver. We found that the Ad vector mediated efficient Pten gene editing even in the presence of typical Ad vector-associated immunotoxicity in the liver. Four months after vector infusion, mice receiving the Pten gene-editing Ad vector showed massive hepatomegaly and features of NASH, consistent with the phenotypes following Cre-loxP-induced Pten deficiency in mouse liver. We also detected induction of humoral immunity against SpCas9 and the potential presence of an SpCas9-specific cellular immune response. Our findings provide a strategy to model human liver diseases in mice and highlight the importance considering Cas9-specific immune responses in future translational studies involving in vivo delivery of CRISPR/Cas9. PMID:26086867

  16. Anti-cas spacers in orphan CRISPR4 arrays prevent uptake of active CRISPR-Cas I-F systems.

    PubMed

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

    2016-01-01

    Archaea and bacteria harbour clustered regularly interspaced short palindromic repeats (CRISPR) loci. These arrays encode RNA molecules (crRNA), each containing a sequence of a single repeat-intervening spacer. The crRNAs guide CRISPR-associated (Cas) proteins to cleave nucleic acids complementary to the crRNA spacer, thus interfering with targeted foreign elements. Notably, pre-existing spacers may trigger the acquisition of new spacers from the target molecule by means of a primed adaptation mechanism. Here, we show that naturally occurring orphan CRISPR arrays that contain spacers matching sequences of the cognate (absent) cas genes are able to elicit both primed adaptation and direct interference against genetic elements carrying those genes. Our findings show the existence of an anti-cas mechanism that prevents the transfer of a fully equipped CRISPR-Cas system. Hence, they suggest that CRISPR immunity may be undesired by particular prokaryotes, potentially because they could limit possibilities for gaining favourable sequences by lateral transfer. PMID:27573106

  17. Observation d'un minimum plat pour RZ Cas [Observation of a flat minimum of RZ Cas

    NASA Astrophysics Data System (ADS)

    Dumont, M.

    1995-07-01

    We observed a minimum of RZ Cas during the night 15/16 august 1991 with the 76 cm telescope of the Fungfraujoch Observatory. We found: Min = HJD 2,448,484.50062 and observed a flat minimum during 10 minutes.

  18. Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9.

    PubMed

    Hirano, Seiichi; Nishimasu, Hiroshi; Ishitani, Ryuichiro; Nureki, Osamu

    2016-03-17

    The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. Our findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9. PMID:26990991

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

    PubMed

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

    2016-03-18

    The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems. PMID:26857072

  20. Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

    PubMed Central

    Gunderson, Felizza F.; Mallama, Celeste A.; Fairbairn, Stephanie G.

    2014-01-01

    Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts. PMID:25547789

  1. BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130Cas

    PubMed Central

    Borre, Pierre Vanden; Near, Richard I.; Makkinje, Anthony; Mostoslavsky, Gustavo; Lerner, Adam

    2011-01-01

    BCAR3 binds to the carboxy-terminus of p130Cas, a focal adhesion adapter protein. Both BCAR3 and p130Cas have been linked to resistance to anti-estrogens in breast cancer, Rac activation and cell motility. Using R743A BCAR3, a point mutant that has lost the ability to bind p130Cas, we find that BCAR3-p130Cas complex formation is not required for BCAR3-mediated anti-estrogen resistance, Rac activation or discohesion of epithelial breast cancer cells. Complex formation was also not required for BCAR3-induced lamellipodia formation in BALB/c-3T3 fibroblasts but was required for optimal BCAR3-induced motility. Although both wildtype and R743A BCAR3 induced phosphorylation of p130Cas and the related adapter protein HEF1/NEDD9, chimeric NSP3:BCAR3 experiments demonstrate that such phosphorylation does not correlate with BCAR3-induced anti-estrogen resistance or lamellipodia formation. Wildtype but not R743A BCAR3 induced lamellipodia formation and augmented cell motility in p130Cas-/- murine embryonic fibroblasts (MEFs), suggesting that while p130Cas itself is not strictly required for these endpoints, complex formation with other CAS family members is, at least in cells lacking p130Cas. Overall, our work suggests that many, but not all, BCAR3-mediated signaling events in epithelial and mesenchymal cells are independent of p130Cas association. These studies also indicate that disruption of the BCAR3-p130Cas complex is unlikely to reverse BCAR3-mediated anti-estrogen resistance. PMID:21262352

  2. An Effective Strategy for Reliably Isolating Heritable and Cas9-Free Arabidopsis Mutants Generated by CRISPR/Cas9-Mediated Genome Editing1[OPEN

    PubMed Central

    Gao, Xiuhua; Chen, Jilin; Dai, Xinhua; Zhang, Da

    2016-01-01

    Mutations generated by CRISPR/Cas9 in Arabidopsis (Arabidopsis thaliana) are often somatic and are rarely heritable. Isolation of mutations in Cas9-free Arabidopsis plants can ensure the stable transmission of the identified mutations to next generations, but the process is laborious and inefficient. Here, we present a simple visual screen for Cas9-free T2 seeds, allowing us to quickly obtain Cas9-free Arabidopsis mutants in the T2 generation. To demonstrate this in principle, we targeted two sites in the AUXIN-BINDING PROTEIN1 (ABP1) gene, whose function as a membrane-associated auxin receptor has been challenged recently. We obtained many T1 plants with detectable mutations near the target sites, but only a small fraction of T1 plants yielded Cas9-free abp1 mutations in the T2 generation. Moreover, the mutations did not segregate in Mendelian fashion in the T2 generation. However, mutations identified in the Cas9-free T2 plants were stably transmitted to the T3 generation following Mendelian genetics. To further simplify the screening procedure, we simultaneously targeted two sites in ABP1 to generate large deletions, which can be easily identified by PCR. We successfully generated two abp1 alleles that contained 1,141- and 711-bp deletions in the ABP1 gene. All of the Cas9-free abp1 alleles we generated were stable and heritable. The method described here allows for effectively isolating Cas9-free heritable CRISPR mutants in Arabidopsis. PMID:27208253

  3. An Effective Strategy for Reliably Isolating Heritable and Cas9-Free Arabidopsis Mutants Generated by CRISPR/Cas9-Mediated Genome Editing.

    PubMed

    Gao, Xiuhua; Chen, Jilin; Dai, Xinhua; Zhang, Da; Zhao, Yunde

    2016-07-01

    Mutations generated by CRISPR/Cas9 in Arabidopsis (Arabidopsis thaliana) are often somatic and are rarely heritable. Isolation of mutations in Cas9-free Arabidopsis plants can ensure the stable transmission of the identified mutations to next generations, but the process is laborious and inefficient. Here, we present a simple visual screen for Cas9-free T2 seeds, allowing us to quickly obtain Cas9-free Arabidopsis mutants in the T2 generation. To demonstrate this in principle, we targeted two sites in the AUXIN-BINDING PROTEIN1 (ABP1) gene, whose function as a membrane-associated auxin receptor has been challenged recently. We obtained many T1 plants with detectable mutations near the target sites, but only a small fraction of T1 plants yielded Cas9-free abp1 mutations in the T2 generation. Moreover, the mutations did not segregate in Mendelian fashion in the T2 generation. However, mutations identified in the Cas9-free T2 plants were stably transmitted to the T3 generation following Mendelian genetics. To further simplify the screening procedure, we simultaneously targeted two sites in ABP1 to generate large deletions, which can be easily identified by PCR. We successfully generated two abp1 alleles that contained 1,141- and 711-bp deletions in the ABP1 gene. All of the Cas9-free abp1 alleles we generated were stable and heritable. The method described here allows for effectively isolating Cas9-free heritable CRISPR mutants in Arabidopsis. PMID:27208253

  4. CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii

    PubMed Central

    Shin, Sung-Eun; Lim, Jong-Min; Koh, Hyun Gi; Kim, Eun Kyung; Kang, Nam Kyu; Jeon, Seungjib; Kwon, Sohee; Shin, Won-Sub; Lee, Bongsoo; Hwangbo, Kwon; Kim, Jungeun; Ye, Sung Hyeok; Yun, Jae-Young; Seo, Hogyun; Oh, Hee-Mock; Kim, Kyung-Jin; Kim, Jin-Soo; Jeong, Won-Joong; Chang, Yong Keun; Jeong, Byeong-ryool

    2016-01-01

    Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including “safe harboring” techniques shown in other organisms. PMID:27291619

  5. Broadening Staphylococcus aureus Cas9 Targeting Range by Modifying PAM Recognition

    PubMed Central

    Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.; Nguyen, Nhu T.; Topkar, Ved V.; Zheng, Zongli; Joung, J. Keith

    2015-01-01

    CRISPR-Cas9 nucleases are primarily guided by RNA-DNA interactions but also require Cas9-mediated recognition of a protospacer adjacent motif (PAM). While potentially advantageous for specificity, extended PAM sequences limit the targeting range of Cas9 orthologues for genome editing. One possible strategy to relieve this restriction is to relax specificities for certain positions within the PAM. Here we used molecular evolution to modify the NNGRRT PAM specificity of Staphylococcus aureus Cas9 (SaCas9). One variant we identified, referred to as KKH SaCas9, shows robust genome editing activities at endogenous human target sites with NNNRRT PAMs. Importantly, using GUIDE-seq, we show that both wild-type and KKH SaCas9 induce comparable numbers of off-target effects in human cells. KKH SaCas9 increased the targeting range of SaCas9 by nearly two- to four-fold. Our molecular evolution strategy does not require structural information and therefore should be applicable to a wide range of Cas9 orthologues. PMID:26524662

  6. Nucleosomes impede Cas9 access to DNA in vivo and in vitro.

    PubMed

    Horlbeck, Max A; Witkowsky, Lea B; Guglielmi, Benjamin; Replogle, Joseph M; Gilbert, Luke A; Villalta, Jacqueline E; Torigoe, Sharon E; Tjian, Robert; Weissman, Jonathan S

    2016-01-01

    The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties. PMID:26987018

  7. Nucleosomes impede Cas9 access to DNA in vivo and in vitro

    PubMed Central

    Horlbeck, Max A; Witkowsky, Lea B; Guglielmi, Benjamin; Replogle, Joseph M; Gilbert, Luke A; Villalta, Jacqueline E; Torigoe, Sharon E; Tjian, Robert; Weissman, Jonathan S

    2016-01-01

    The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties. DOI: http://dx.doi.org/10.7554/eLife.12677.001 PMID:26987018

  8. Force Sensing by Extension of the Src Family Kinase Substrate, p130Cas

    PubMed Central

    Sawada, Yasuhiro; Tamada, Masako; Dubin-Thaler, Benjamin J.; Cherniavskaya, Oksana; Sakai, Ryuichi; Tanaka, Sakae; Sheetz, Michael P.

    2009-01-01

    SUMMARY Physical force is implicated in many cell functions. However, the molecular mechanisms of force sensing are poorly understood. Here, we show that mechanical extension of p130Cas (Cas) in vitro and in vivo causes phosphorylation by Src family kinases with no apparent change in Src kinase activity and that Cas phosphorylation is involved in stretch-dependent activation of the small GTPase, Rap1. In vitro, we mechanically extended bacterially expressed Cas substrate domain (CasSD) and found a remarkable enhancement of phosphorylation by specific kinases. Using an antibody that recognized extended CasSD in vitro, Cas extension in intact cells was observed in the peripheral regions of spreading cells where higher traction forces are expected and phosphorylated Cas was detected, suggesting that the in vitro extension and phosphorylation of CasSD is relevant to physiological force transduction. Thus, Cas acts as a primary force-sensor through extension of the substrate domain, which primes it for phosphorylation. PMID:17129785

  9. CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii.

    PubMed

    Shin, Sung-Eun; Lim, Jong-Min; Koh, Hyun Gi; Kim, Eun Kyung; Kang, Nam Kyu; Jeon, Seungjib; Kwon, Sohee; Shin, Won-Sub; Lee, Bongsoo; Hwangbo, Kwon; Kim, Jungeun; Ye, Sung Hyeok; Yun, Jae-Young; Seo, Hogyun; Oh, Hee-Mock; Kim, Kyung-Jin; Kim, Jin-Soo; Jeong, Won-Joong; Chang, Yong Keun; Jeong, Byeong-Ryool

    2016-01-01

    Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including "safe harboring" techniques shown in other organisms. PMID:27291619

  10. Dissociation of CAS phase in the uppermost lower mantle

    NASA Astrophysics Data System (ADS)

    Ishibashi, Kazufusa; Hirose, Kei; Sata, Nagayoshi; Ohishi, Yasuo

    2008-05-01

    The high-pressure stability limit of calcium aluminosilicate (CAS) phase has been examined in its end-member CaAl4Si2O11 composition at 18 39 GPa and 1,670 2,300 K in a laser-heated diamond-anvil cell (LHDAC). The in-situ synchrotron X-ray diffraction measurements revealed that the CAS phase decomposes into three-phase assemblage of cubic Al-bearing CaSiO3 perovskite, Al2O3 corundum, and SiO2 stishovite above 30 GPa and 2,000 K with a positive pressure temperature slope. Present results have important implications for the subsolidus mineral assemblage of subducted sediment and the melting phase relation of basalt in the lower mantle.

  11. Chromosome engineering in zygotes with CRISPR/Cas9

    PubMed Central

    Boroviak, Katharina; Doe, Brendan; Banerjee, Ruby; Yang, Fengtang

    2016-01-01

    SUMMARY Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection. genesis 54:78–85, 2016. © 2016 The Authors. genesis Published by Wiley Periodicals, Inc. PMID:26742453

  12. Next Generation Prokaryotic Engineering: The CRISPR-Cas Toolkit.

    PubMed

    Mougiakos, Ioannis; Bosma, Elleke F; de Vos, Willem M; van Kranenburg, Richard; van der Oost, John

    2016-07-01

    The increasing demand for environmentally friendly production processes of green chemicals and fuels has stimulated research in microbial metabolic engineering. CRISPR-Cas-based tools for genome editing and expression control have enabled fast, easy, and accurate strain development for established production platform organisms, such as Escherichia coli and Saccharomyces cerevisiae. However, the growing interest in alternative production hosts, for which genome editing options are generally limited, requires further developing such engineering tools. In this review, we discuss established and emerging CRISPR-Cas-based tools for genome editing and transcription control of model and non-model prokaryotes, and we analyse the possibilities for further improvement and expansion of these tools for next generation prokaryotic engineering. PMID:26944793

  13. Photometry of stars in the Cas OB5 Associations

    NASA Astrophysics Data System (ADS)

    Tanriver, Mehmet; Keskin, Ahmet

    2016-07-01

    OB associations are a grouping of very young associations, contain 10-100 very hot massive stars, spectral types O and B. Also, the OB associations contain low and intermediate mass stars, too. Association members are believed to form within the same small volume inside a giant molecular cloud. Once the surrounding dust and gas is blown away, the remaining stars become not tied up and begin to drift separately. It is believed that the majority of all stars in the Milky Way were formed in OB associations. O type stars are short-lived, and will be at an end as supernovae after roundly a million years. OB associations are generally only a few million years in age or less. In this study, the photometry of UU Cas and field star which been Cas OB5 association member was carried out. Light curves and color diagrams are given in the study.

  14. Highly efficient targeted chromosome deletions using CRISPR/Cas9.

    PubMed

    He, Zuyong; Proudfoot, Chris; Mileham, Alan J; McLaren, David G; Whitelaw, C Bruce A; Lillico, Simon G

    2015-05-01

    The CRISPR/Cas9 system has emerged as an intriguing new technology for genome engineering. It utilizes the bacterial endonuclease Cas9 which, when delivered to eukaryotic cells in conjunction with a user-specified small guide RNA (gRNA), cleaves the chromosomal DNA at the target site. Here we show that concurrent delivery of gRNAs designed to target two different sites in a human chromosome introduce DNA double-strand breaks in the chromosome and give rise to targeted deletions of the intervening genomic segment. Predetermined genomic DNA segments ranging from several-hundred base pairs to 1 Mbp can be precisely deleted at frequencies of 1-10%, with no apparent correlation between the size of the deleted fragment and the deletion frequency. The high efficiency of this technique holds promise for large genomic deletions that could be useful in generation of cell and animal models with engineered chromosomes. PMID:25362885

  15. Energy biotechnology in the CRISPR-Cas9 era.

    PubMed

    Estrela, Raissa; Cate, Jamie Harrison Doudna

    2016-04-01

    The production of bioenergy from plant biomass previously relied on using microorganisms that rapidly and efficiently convert simple sugars into fuels and chemicals. However, to exploit the far more abundant carbon fixed in plant cell walls, future industrial production hosts will need to be engineered to leverage the most efficient biochemical pathways and most robust traits that can be found in nature. The CRISPR-Cas9 genome editing technology now enables writing the genome at will, which will allow biotechnology to become an 'information science.' This review covers recent advances in using CRISPR-Cas9 to engineer the genomes of a wide variety of organisms that could be use in the industrial production of biofuels and renewable chemicals. PMID:26874259

  16. Application of CRISPR/Cas9 Technology to HBV

    PubMed Central

    Lin, Guigao; Zhang, Kuo; Li, Jinming

    2015-01-01

    More than 240 million people around the world are chronically infected with hepatitis B virus (HBV). Nucleos(t)ide analogs and interferon are the only two families of drugs to treat HBV currently. However, none of these anti-virals directly target the stable nuclear covalently closed circular DNA (cccDNA), which acts as a transcription template for viral mRNA and pre-genomic RNA synthesis and secures virus persistence. Thus, the fact that only a small number of patients treated achieve sustained viral response (SVR) or cure, highlights the need for new therapies against HBV. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system can specifically target the conserved regions of the HBV genome. This results in robust viral suppression and provides a promising tool for eradicating the virus. In this review, we discuss the function and application of the CRISPR/Cas9 system as a novel therapy for HBV. PMID:26540039

  17. Plasmocytome costal solitaire: à propos d'un cas

    PubMed Central

    Razafimanjato, Narindra Njarasoa Mihaja; Ravoatrarilandy, Manjakaniaina; Rakotoarisoa, Andriamihaja Jean Claude; Hasiniatsy, Rodrigue; Hunald, Allen Francis; Rakototiana, Auberlin Felantsoa; Rafaramino, Florine; Rakotovao, Hanitrala Jean Louis

    2014-01-01

    Les auteurs rapportent un cas de plasmocytome solitaire particulière par leur localisation costale. Le diagnostic est basé sur la mise en évidence d'une tumeur localisée, constituée de cellules plasmocytaires monoclonales cytologiquement identiques à celles du myélome multiple, en l'absence d'autres signes en faveur d'une forme disséminée. Nous rapportons un cas de plasmocytome solitaire à localisation costale et nous discutons les aspects diagnostiques et thérapeutiques de cette affection potentiellement menacée dans son évolution par la transformation en myélome multiple. PMID:25419306

  18. Analysis of estimation algorithms for CDTI and CAS applications

    NASA Technical Reports Server (NTRS)

    Goka, T.

    1985-01-01

    Estimation algorithms for Cockpit Display of Traffic Information (CDTI) and Collision Avoidance System (CAS) applications were analyzed and/or developed. The algorithms are based on actual or projected operational and performance characteristics of an Enhanced TCAS II traffic sensor developed by Bendix and the Federal Aviation Administration. Three algorithm areas are examined and discussed. These are horizontal x and y, range and altitude estimation algorithms. Raw estimation errors are quantified using Monte Carlo simulations developed for each application; the raw errors are then used to infer impacts on the CDTI and CAS applications. Applications of smoothing algorithms to CDTI problems are also discussed briefly. Technical conclusions are summarized based on the analysis of simulation results.

  19. Inactivation of Cancer Mutations Utilizing CRISPR/Cas9.

    PubMed

    Gebler, Christina; Lohoff, Tim; Paszkowski-Rogacz, Maciej; Mircetic, Jovan; Chakraborty, Debojyoti; Camgoz, Aylin; Hamann, Martin V; Theis, Mirko; Thiede, Christian; Buchholz, Frank

    2017-01-01

    Although whole-genome sequencing has uncovered a large number of mutations that drive tumorigenesis, functional ratification for most mutations remains sparse. Here, we present an approach to test functional relevance of tumor mutations employing CRISPR/Cas9. Combining comprehensive sgRNA design and an efficient reporter assay to nominate efficient and selective sgRNAs, we establish a pipeline to dissect roles of cancer mutations with potential applicability to personalized medicine and future therapeutic use. PMID:27576906

  20. Targeted mutagenesis using CRISPR/Cas system in medaka

    PubMed Central

    Ansai, Satoshi; Kinoshita, Masato

    2014-01-01

    ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-based RNA-guided endonuclease (RGEN) has recently emerged as a simple and efficient tool for targeted genome editing. In this study, we showed successful targeted mutagenesis using RGENs in medaka, Oryzias latipes. Somatic and heritable mutations were induced with high efficiency at the targeted genomic sequence on the DJ-1 gene in embryos that had been injected with the single guide RNA (sgRNA) transcribed by a T7 promoter and capped RNA encoding a Cas9 nuclease. The sgRNAs that were designed for the target genomic sequences without the 5′ end of GG required by the T7 promoter induced the targeted mutations. This suggests that the RGEN can target any sequence adjacent to an NGG protospacer adjacent motif (PAM) sequence, which occurs once every 8 bp. The off-target alterations at 2 genomic loci harboring double mismatches in the 18-bp targeting sequences were induced in the RGEN-injected embryos. However, we also found that the off-target effects could be reduced by lower dosages of sgRNA. Taken together, our results suggest that CRISPR/Cas-mediated RGENs may be an efficient and flexible tool for genome editing in medaka. PMID:24728957

  1. Embolie de liquide amniotique: à propos de deux cas

    PubMed Central

    Elbahraoui, Houda; Bouziane, Hanane; Elghanmi, Adil; Lakhdar, Amina; Elhanchi, Zaki; Ferhati, Driss

    2012-01-01

    L’embolie de liquide amniotique (ELA) est une complication imprévisible de l’accouchement, souvent fatale, associant un collapsus cardiovasculaire sévère, un syndrome de détresse respiratoire aiguë et une hémorragie avec coagulation intra vasculaire disséminée (CIVD). Dès l’évocation du diagnostic, la prise en charge doit être multidisciplinaire et intensive. ELA est responsable d’une mortalité maternelle et néonatale importante, son incidence est extrêmement variable selon les études et le taux de mortalité maternelle varie entre 26 et 86 % selon les études. Ces dix dernières années, le pronostic materno-fœtal semble en amélioration grâce aux progrès de prise en charge standardisée multidisciplinaire sur les lieux d’accouchement. Nous rapportons deux cas d’embolie de liquide amniotique. Le premier cas s’est manifesté au cours du travail et le deuxième cas est survenu dans les suites immédiates de l’accouchement. PMID:22655108

  2. Motions of alloying additions in the CAS steelmaking operations

    NASA Astrophysics Data System (ADS)

    Mazumdar, D.; Guthrie, R. I. L.

    1993-08-01

    Water model studies in a pilot scale ladle ( D = 1.12 m and L = 0.93 m) were carried out to investigate the subsurface motion of both buoyant and sinking additions during the CAS (com-position adjustment by sealed argon bubbling systems) alloy addition procedure in steelmaking. This technique involves placing a refractory baffle around a rising gas/liquid plume within a stirred ladle of steel. Alloy additions are then made by projecting them into the slag-free region of steel within the baffled region. It was found that such particles while moving through the upwelling two-phase plume region can experience a significant reduction in drag forces, causing buoyant particles to penetrate more deeply than anticipated for a homogeneous fluid. Therefore, considering reduced drag on particles penetrating the upwelling gas liquid plume region, predictions were made for the trajectories of spherical-shaped particles using Newton’s law of motion. Predictions were in very reasonable agreement with those measured. Incorporating the velocity fields in industrial size vessels already reported by the present authors, trajectories of spherical-shaped additions (diameter ˜ 80 mm) in a 150-ton ladle during CAS operations were then predicted. The industrial implications of such trajectories, together with the alloy’s dissolution and dispersion behavior, were also analyzed. Finally, advantages of the CAS alloy addition procedure over conventional methods, in terms of the recovery rates of buoyant additions, are discussed.

  3. CRISPR-Cas systems for genome editing, regulation and targeting

    PubMed Central

    Sander, Jeffry D.; Joung, J. Keith

    2014-01-01

    Targeted genome editing using engineered nucleases has rapidly transformed from a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered regularly interspaced short palindromic repeat (CRISPR) technology, an important new platform for generating RNA-guided nucleases (RGNs), such as Cas9, with customizable specificities. RGN-mediated genome editing is facile, rapid and has enabled the efficient modification of endogenous genes in a wide variety of biomedically important cell types and novel organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the capabilities of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease. PMID:24584096

  4. Generation of an Oocyte-Specific Cas9 Transgenic Mouse for Genome Editing

    PubMed Central

    Zhang, Linlin; Zhou, Jiankui; Han, Jinxiong; Hu, Bian; Hou, Ningning; Shi, Yun; Huang, Xingxu

    2016-01-01

    The CRISPR/Cas9 system has been developed as an easy-handle and multiplexable approach for engineering eukaryotic genomes by zygote microinjection of Cas9 and sgRNA, while preparing Cas9 for microinjection is laborious and introducing inconsistency into the experiment. Here, we describe a modified strategy for gene targeting through using oocyte-specific Cas9 transgenic mouse. With this mouse line, we successfully achieve precise gene targeting by injection of sgRNAs only into one-cell-stage embryos. Through comprehensive analysis, we also show allele complexity and off-target mutagenesis induced by this strategy is obviously lower than Cas9 mRNA/sgRNA injection. Thus, injection of sgRNAs into oocyte-specific Cas9 transgenic mouse embryo provides a convenient, efficient and reliable approach for mouse genome editing. PMID:27119535

  5. Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein.

    PubMed

    Silas, Sukrit; Mohr, Georg; Sidote, David J; Markham, Laura M; Sanchez-Amat, Antonio; Bhaya, Devaki; Lambowitz, Alan M; Fire, Andrew Z

    2016-02-26

    CRISPR systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in type I and II CRISPR systems by the acquisition of short segments of DNA (spacers) from invasive elements. In several type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we showed that a RT-Cas1 fusion protein enables the acquisition of RNA spacers in vivo in a RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze the ligation of RNA segments into the CRISPR array, which is followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. PMID:26917774

  6. Evidence for the widespread distribution of CRISPR-Cas system in the Phylum Cyanobacteria

    PubMed Central

    Cai, Fei; Axen, Seth D.; Kerfeld, Cheryl A.

    2013-01-01

    Members of the phylum Cyanobacteria inhabit ecologically diverse environments. However, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR associated genes), an extremely adaptable defense system, has not been surveyed in this phylum. We analyzed 126 cyanobacterial genomes and, surprisingly, found CRISPR-Cas in the majority except the marine subclade (Synechococcus and Prochlorococcus), in which cyanophages are a known force shaping their evolution. Multiple observations of CRISPR loci in the absence of cas1/cas2 genes may represent an early stage of losing a CRISPR-Cas locus. Our findings reveal the widespread distribution of their role in the phylum Cyanobacteria and provide a first step to systematically understanding CRISPR-Cas systems in cyanobacteria. PMID:23628889

  7. Transcriptional Regulation with CRISPR/Cas9 Effectors in Mammalian Cells.

    PubMed

    Pham, Hannah; Kearns, Nicola A; Maehr, René

    2016-01-01

    CRISPR/Cas9-based regulation of gene expression provides the scientific community with a new high-throughput tool to dissect the role of genes in molecular processes and cellular functions. Single-guide RNAs allow for recruitment of a nuclease-dead Cas9 protein and transcriptional Cas9-effector fusion proteins to specific genomic loci, thereby modulating gene expression. We describe the application of a CRISPR-Cas9 effector system from Streptococcus pyogenes for transcriptional regulation in mammalian cells resulting in activation or repression of transcription. We present methods for appropriate target site selection, sgRNA design, and delivery of dCas9 and dCas9-effector system components into cells through lentiviral transgenesis to modulate transcription. PMID:26463376

  8. Long-term monitoring of orbital modulation and secondary-star irradiation in Nova Cas 1995 (V723 Cas)

    NASA Astrophysics Data System (ADS)

    Ochner, P.; Moschini, F.; Munari, U.; Frigo, A.

    2015-11-01

    We present optical spectroscopy collected at seven epochs and BVRCIC photometry obtained at 1227 epochs of nova V723 Cas, covering the time interval between 2007 and 2015. The mean magnitude during this period, stable at ˜3 mag brighter than in quiescence, and the continuous presence of strong [Fe X] and other high-ionization emission lines, indicates that the nuclear burning at the surface of the white dwarf is continuing 20 years past the initial outburst. The light curve shows a large amplitude (2 mag) orbital modulation, which is governed by the visibility of the irradiated side of the secondary star. Our observations do not confirm the reported increase with time of the orbital period of V723 Cas, a period of P=16.638 383 ± 0.000 025 h satisfying equally well all available observations in all bands. Our observations also do not confirm the presence of an additional periodicity around P=15.2397 h from which V723 Cas was classified as an intermediate-polar system.

  9. Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases.

    PubMed Central

    Vuori, K; Hirai, H; Aizawa, S; Ruoslahti, E

    1996-01-01

    Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. Among these are the focal adhesion proteins p130cas (Cas) and focal adhesion kinase (FAK). Here we identify the kinase(s) mediating integrin-induced Cas phosphorylation and characterize protein-protein interactions mediated by phosphorylated Cas. We found that expression of a constitutively active FAK in fibroblasts results in a consecutive tyrosine phosphorylation of Cas. This effect required the autophosphorylation site of FAK, which is a binding site for Src family kinases. Integrin-mediated phosphorylation of Cas was not, however, compromised in fibroblasts lacking FAK. In contrast, adhesion-induced tyrosine phosphorylation of Cas was reduced in cells lacking Src, whereas enhanced phosphorylation of Cas was observed Csk- cells, in which Src kinases are activated. These results suggest that Src kinases are responsible for the integrin-mediated tyrosine phosphorylation of Cas. FAK seems not to be necessary for phosphorylation of Cas, but when autophosphorylated, FAK may recruit Src family kinases to phosphorylate Cas. Cas was found to form complexes with Src homology 2 (SH2) domain-containing signaling molecules, such as the SH2/SH3 adapter protein Crk, following integrin-induced tyrosine phosphorylation. Guanine nucleotide exchange factors C3G and Sos were found in the Cas-Crk complex upon integrin ligand binding. These observations suggest that Cas serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion. PMID:8649368

  10. Efficient Production of Gene-Modified Mice using Staphylococcus aureus Cas9

    PubMed Central

    Zhang, Xiya; Liang, Puping; Ding, Chenhui; Zhang, Zhen; Zhou, Jianwen; Xie, Xiaowei; Huang, Rui; Sun, Ying; Sun, Hongwei; Zhang, Jinran; Xu, Yanwen; Songyang, Zhou; Huang, Junjiu

    2016-01-01

    The CRISPR/Cas system is an efficient genome-editing tool to modify genes in mouse zygotes. However, only the Streptococcus pyogenes Cas9 (SpCas9) has been systematically tested for generating gene-modified mice. The protospacer adjacent motif (PAM, 5′-NGG-3′) recognized by SpCas9 limits the number of potential target sites for this system. Staphylococcus aureus Cas9 (SaCas9), with its smaller size and unique PAM (5′-NNGRRT-3′) preferences, presents an alternative for genome editing in zygotes. Here, we showed that SaCas9 could efficiently and specifically edit the X-linked gene Slx2 and the autosomal gene Zp1 in mouse zygotes. SaCas9-mediated disruption of the tyrosinase (Tyr) gene led to C57BL/6J mice with mosaic coat color. Furthermore, multiplex targeting proved efficient multiple genes disruption when we co-injected gRNAs targeting Slx2, Zp1, and Tyr together with SaCas9 mRNA. We were also able to insert a Flag tag at the C-terminus of histone H1c, when a Flag-encoding single-stranded DNA oligo was co-introduced into mouse zygotes with SaCas9 mRNA and the gRNA. These results indicate that SaCas9 can specifically cleave the target gene locus, leading to successful gene knock-out and precise knock-in in mouse zygotes, and highlight the potential of using SaCas9 for genome editing in preimplantation embryos and producing gene-modified animal models. PMID:27586692