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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Journal ArticleDOI
Advanced Good Cell Culture Practice for Human Primary, Stem Cell-Derived and Organoid Models as well as Microphysiological Systems
David Pamies,Anna Bal-Price,Christophe Chesne,Sandra Coecke,Andras Dinnyes,Chantra Eskes,Regina Grillari,Gerhard Gstraunthaler,Thomas Hartung,Thomas Hartung,Paul Jennings,Marcel Leist,Ulrich Martin,Robert Passier,Jens Christian Schwamborn,Glyn Stacey,Heidrun Ellinger-Ziegelbauer,Mardas Daneshian +17 more
TL;DR: Recent key developments in in vitro cell culture are summarized and addressed and the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells are addressed.
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Gene Drive for Mosquito Control: Where Did It Come from and Where Are We Headed?
TL;DR: The lengthy history of genetic advances in mosquito biology is reviewed and both the impact of efficient site-specific gene editing on vector biology and the resulting potential to deploy new genetic tools for the abatement of mosquito-borne disease are discussed.
Journal ArticleDOI
High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells.
Hui Kwon Kim,Sungtae Lee,Younggwang Kim,Jinman Park,Seonwoo Min,Jae Woo Choi,Tony P. Huang,Tony P. Huang,Tony P. Huang,Sungroh Yoon,David R. Liu,David R. Liu,David R. Liu,Hyongbum Kim +13 more
TL;DR: An extensive comparison of the PAM-sequence compatibilities and the on-target and off-target activities of Cas9 from Streptococcus pyogenes (SpCas9) and the Sp Cas9 variants xCas9 and SpCas9-NG and it is found that x Cas9 has the lowest tolerance for mismatched target sequences and that SpCas 9-NG has the broadest PAM compatibility.
Journal ArticleDOI
CRISPR/Cas9 for Cancer Therapy: Hopes and Challenges
Marta Martinez-Lage,Pilar Puig-Serra,Pablo Menendez,Pablo Menendez,Raúl Torres-Ruiz,Sandra Rodriguez-Perales +5 more
TL;DR: Important considerations for the use of CRISPR/Cas9 in therapeutic settings and major challenges that will need to be addressed prior to its clinical translation for a complex and polygenic disease such as cancer are discussed.
Patent
Cas variants for gene editing
David R. Liu,Alexis C. Komor +1 more
TL;DR: In this paper, the authors present strategies, systems, reagents, methods and kits for targeted nucleic acid editing, including editing a single site within the genome of a cell or subject, eg, within the human genome.
References
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Journal ArticleDOI
Geneious Basic
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.
Journal ArticleDOI
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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