Rationally engineered Cas9 nucleases with improved specificity
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...
Main Authors: | , , , , , |
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Other Authors: | , , , |
Format: | Article |
Language: | English |
Published: |
American Association for the Advancement of Science (AAAS),
2016-05-22T23:25:15Z.
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Subjects: | |
Online Access: | Get fulltext |
Summary: | 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. Massachusetts Institute of Technology. Simons Center for the Social Brain National Institute of General Medical Sciences (U.S.) (T32GM007753) Paul & Daisy Soros Fellowships for New Americans (New York, N.Y.) National Institute of Mental Health (U.S.) (5DP1-MH100706) National Institute of Mental Health (U.S.) (1R01MH110049) National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (5R01DK097768-03) National Science Foundation (U.S.) (Waterman Award) W. M. Keck Foundation New York Stem Cell Foundation Damon Runyon Cancer Research Foundation Kinship Foundation. Searle Scholars Program Merkin Foundation Vallee Foundation |
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