Rationally engineered Cas9 nucleases with improved specificity

• Main Authors: Slaymaker, Ian (Contributor), Gao, Linyi (Contributor), Zetsche, Bernd (Contributor), Scott, David Arthur (Contributor), Yan, Winston Xia (Contributor), Zhang, Feng (Contributor)
• Other Authors: Harvard University- (Contributor), Massachusetts Institute of Technology. Department of Biological Engineering (Contributor), Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences (Contributor), McGovern Institute for Brain Research at MIT (Contributor)
• Format: Article
• Language: English
• Published: American Association for the Advancement of Science (AAAS), 2016-05-22T23:25:15Z.
• Subjects: Article
• Online Access: Get fulltext

 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.