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One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering
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TLDR
In this article, the type II bacterial CRISPR/Cas system is used to create mice with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes.Abstract:
The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.read more
Citations
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Journal ArticleDOI
Genome engineering using the CRISPR-Cas9 system
F. Ann Ran,Patrick D. Hsu,Jason Wright,Vineeta Agarwala,Vineeta Agarwala,David A. Scott,Feng Zhang +6 more
TL;DR: A set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies are described.
Journal ArticleDOI
The new frontier of genome engineering with CRISPR-Cas9
TL;DR: The power of the CRISPR-Cas9 technology to systematically analyze gene functions in mammalian cells, study genomic rearrangements and the progression of cancers or other diseases, and potentially correct genetic mutations responsible for inherited disorders is illustrated.
Development and Applications of CRISPR-Cas9 for Genome Engineering
TL;DR: The development and applications of Cas9 are described for a variety of research or translational applications while highlighting challenges as well as future directions.
Book ChapterDOI
Genome engineering using CRISPR-Cas9 system.
Le Cong,Feng Zhang +1 more
TL;DR: This chapter presents all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination.
Journal ArticleDOI
Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation
John G. Doench,Ella Hartenian,Daniel B. Graham,Zuzana Tothova,Mudra Hegde,Ian Smith,Meagan E Sullender,Benjamin L. Ebert,Ramnik J. Xavier,David E. Root +9 more
TL;DR: An online tool for the design of highly active sgRNAs for any gene of interest is provided, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9.
References
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Journal ArticleDOI
A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
Martin Jinek,Krzysztof Chylinski,Krzysztof Chylinski,Ines Fonfara,Michael H. Hauer,Jennifer A. Doudna,Emmanuelle Charpentier +6 more
TL;DR: This study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,F. A. Ran,David Benjamin Turitz Cox,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +10 more
TL;DR: Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
Journal ArticleDOI
RNA-Guided Human Genome Engineering via Cas9
Prashant Mali,Luhan Yang,Kevin M. Esvelt,John Aach,Marc Güell,James E. DiCarlo,Julie E. Norville,George M. Church,George M. Church +8 more
TL;DR: The type II bacterial CRISPR system is engineer to function with custom guide RNA (gRNA) in human cells to establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
Journal ArticleDOI
Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.
Lei S. Qi,Matthew H. Larson,Luke A. Gilbert,Jennifer A. Doudna,Jonathan S. Weissman,Adam P. Arkin,Adam P. Arkin,Wendell A. Lim +7 more
TL;DR: This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale and can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects.
DNA targeting specificity of RNA-guided Cas9 nucleases
Patrick D. Hsu,David A. Scott,David A. Scott,Joshua A. Weinstein,Joshua A. Weinstein,F. Ann Ran,F. Ann Ran,F. Ann Ran,Silvana Konermann,Silvana Konermann,Vineeta Agarwala,Vineeta Agarwala,Vineeta Agarwala,Yinqing Li,Yinqing Li,Eli J. Fine,Xuebing Wu,Ophir Shalem,Ophir Shalem,Thomas J. Cradick,Luciano A. Marraffini,Gang Bao,Feng Zhang,Feng Zhang +23 more
TL;DR: It is found that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches.