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- Title
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.
- Authors
Tsai, Shengdar Q; Zheng, Zongli; Nguyen, Nhu T; Liebers, Matthew; Topkar, Ved V; Thapar, Vishal; Wyvekens, Nicolas; Khayter, Cyd; Iafrate, A John; Le, Long P; Aryee, Martin J; Joung, J Keith
- Abstract
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.
- Subjects
GENOME editing; NUCLEASES; DNA damage; IMMUNOPRECIPITATION; CELL lines
- Publication
Nature Biotechnology, 2015, Vol 33, Issue 2, p187
- ISSN
1087-0156
- Publication type
Article
- DOI
10.1038/nbt.3117