期刊
NATURE BIOTECHNOLOGY
卷 33, 期 2, 页码 187-197出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.3117
关键词
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资金
- National Institutes of Health (NIH) Director's Pioneer Award [DP1 GM105378]
- NIH [R01 GM088040, R01 AR063070, F32 GM105189]
- Jim and Ann Orr Massachusetts General Hospital (MGH) Research Scholar Award
- US Army Research Laboratory
- US Army Research Office [W911NF-11-2-0056]
CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced, off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases before clinical use.
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