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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.

Author information

1
Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.
2
Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.
3
Department of Pathology, Harvard Medical School, Boston, MA USA.
4
Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
#
Contributed equally

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.

Comment in

PMID:
25513782
PMCID:
PMC4320685
DOI:
10.1038/nbt.3117
[Indexed for MEDLINE]
Free PMC Article

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