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Nat Biotechnol. 2015 Feb;33(2):187-197. doi: 10.1038/nbt.3117. Epub 2014 Dec 16.

GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.

Tsai SQ#1,2,3, Zheng Z#1,2,3,4, Nguyen NT1,2, Liebers M1,2, Topkar VV1,2, Thapar V1,2, Wyvekens N1,2, Khayter C1,2, Iafrate AJ1,2,3, Le LP1,2,3, Aryee MJ1,2,3, Joung JK1,2,3.

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Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.
Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.
Department of Pathology, Harvard Medical School, Boston, MA USA.
Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
Contributed equally


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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