期刊
SCIENCE
卷 351, 期 6268, 页码 84-88出版社
AMER ASSOC ADVANCEMENT SCIENCE
DOI: 10.1126/science.aad5227
关键词
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资金
- Simons Center for the Social Brain
- National Institute of General Medical Sciences [T32GM007753]
- Paul and Daisy Soros Fellowship
- National Institutes of Health through NIMH [5DP1-MH100706, 1R01MH110049]
- NIDDK [5R01DK097768-03]
- National Science Foundation
- Keck Foundation
- New York Stem Cell Foundation
- Damon Runyon Foundation
- Searle Scholars Foundation
- Merkin Foundation
- Vallee Foundation
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [R01DK097768] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [T32GM007753, T32GM008313] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF MENTAL HEALTH [DP1MH100706, R01MH110049] Funding Source: NIH RePORTER
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.
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