• Shengdar Q Tsai, Zongli Zheng, Nhu T Nguyen, Matthew Liebers, Ved V Topkar, Vishal Thapar, Nicolas Wyvekens, Cyd Khayter, A John Iafrate, Long P Le, Martin J Aryee, and J Keith Joung.
• 1] Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [2] Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [3] Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
• Nat. Biotechnol. 2015 Feb 1; 33 (2): 187-97.

AbstractCRISPR 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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