Tsai, Shengdar Q.Zheng, ZongliNguyen, Nhu T.Liebers, MatthewTopkar, VedThapar, VishalWyvekens, NicolasKhayter, CydIafrate, A. JohnLe, Long P.Aryee, MartinJoung, J. Keith2015-09-012014Tsai, S. Q., Z. Zheng, N. T. Nguyen, M. Liebers, V. V. Topkar, V. Thapar, N. Wyvekens, et al. 2014. “GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.” Nature biotechnology 33 (2): 187-197. doi:10.1038/nbt.3117. http://dx.doi.org/10.1038/nbt.3117.1087-0156http://nrs.harvard.edu/urn-3:HUL.InstRepos:21460330CRISPR 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 breaks Application of GUIDE-Seq to thirteen 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 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 prior to clinical use.en-USJournal Article2015-09-0110.1038/nbt.3117