期刊
NATURE BIOTECHNOLOGY
卷 31, 期 9, 页码 822-+出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.2623
关键词
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资金
- US National Institutes of Health (NIH) [DP1 GM105378, NIH R01 GM088040, NIH P50 HG005550]
- Defense Advanced Research Projects Agency (DARPA) [W911NF-11-2-0056]
- Jim and Ann Orr Massachusetts General Hospital Research Scholar Award
Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell-based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas) 9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.
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