期刊
NATURE
卷 529, 期 7587, 页码 490-+出版社
NATURE PORTFOLIO
DOI: 10.1038/nature16526
关键词
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资金
- Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship
- Massachusetts General Hospital (MGH) Department of Pathology
- MGH Tosteson
- Fund for Medical Discovery Fellowship
- US National Institutes of Health (NIH) Director's Pioneer Award [DP1 GM105378]
- NIH [R01 GM107427, R01 GM088040]
- Jim and Ann Orr MGH Research Scholar Award
CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with > 85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.
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