Rationally engineered Cas9 nucleases with improved specificity

Slaymaker, Ian M.; Gao, Linyi; Zetsche, Bernd; Scott, David; Yan, Winston X.; Zhang, Feng

doi:10.1126/science.aad5227

articleScienceDec 1, 2015Closed access

Rationally engineered Cas9 nucleases with improved specificity

IMIan M. Slaymaker LGLinyi Gao BZBernd Zetsche DSDavid Scott WXWinston X. Yan

Broad Institute · McGovern Institute for Brain Research · +4 more institutions

PubMed

Indexed incrossrefpubmed

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…

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2,507

total citations

FWCI: 118.50
Percentile: 100%
References: 22

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Topics & keywords

Topics

Keywords

CRISPR
Cas9
Genome editing
Computational biology
Genome engineering
Cleavage (geology)
DNA
Streptococcus pyogenes

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