Rationally Engineered Cas9 Nucleases With Improved Specificity
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TLDR
In this paper, the authors used structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9) using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells.Abstract:
The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.read more
Citations
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The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex.
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TL;DR: Examining the DNA cleavage activities of Streptococcus pyogenes Cas9 and its mutants using various target sequences and study the conformational dynamics of R-loop structure during target binding using single-molecule fluorescence energy transfer (smFRET) technique provide new insights into the process ofR-loop formation.
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Class 2 CRISPR-Cas RNA-guided endonucleases: Swiss Army knives of genome editing.
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Distinct patterns of Cas9 mismatch tolerance in vitro and in vivo
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References
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Matthew Kearse,Richard Moir,Amy Wilson,Steven Stones-Havas,Matthew Cheung,Shane Sturrock,Simon Buxton,Alex Cooper,Sidney Markowitz,Chris Duran,Tobias Thierer,Bruce Ashton,Peter Meintjes,Alexei J. Drummond +13 more
TL;DR: Geneious Basic has been designed to be an easy-to-use and flexible desktop software application framework for the organization and analysis of biological data, with a focus on molecular sequences and related data types.
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Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,Le Cong,F. Ann Ran,F. Ann Ran,David M. Cox,David M. Cox,Shuailiang Lin,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +15 more
TL;DR: The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage as discussed by the authors.
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
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: In this article, the Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing.
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