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


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.

SUBMITTER: Tsai SQ 

PROVIDER: S-EPMC4320685 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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

Tsai Shengdar Q SQ   Zheng Zongli Z   Nguyen Nhu T NT   Liebers Matthew M   Topkar Ved V VV   Thapar Vishal V   Wyvekens Nicolas N   Khayter Cyd C   Iafrate A John AJ   Le Long P LP   Aryee Martin J MJ   Joung J Keith JK  

Nature biotechnology 20141216 2


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-  ...[more]

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