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

Nat Biotechnol 2015 Feb 16;33(2):187-197. Epub 2014 Dec 16.

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.

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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http://dx.doi.org/10.1038/nbt.3117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320685PMC
February 2015
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