Details:

Alternative Title:
Nat Biotechnol
Personal Author:
Tsai, Shengdar Q. ; Zheng, Zongli ; Nguyen, Nhu T. ; Liebers, Matthew ; Topkar, Ved V. ; ... More +
Tsai, Shengdar Q. ; Zheng, Zongli ; Nguyen, Nhu T. ; Liebers, Matthew ; Topkar, Ved V. ; Thapar, Vishal ; Wyvekens, Nicolas ; Khayter, Cyd ; Iafrate, A. John ; Le, Long P. ; Aryee, Martin J. ; Joung, J. Keith ; Less -
Description:
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.
Subject:
[+]
Article
Cell Line
CRISPR-Cas Systems
DNA Breaks, Double-Stranded
Genome, Human
High-Throughput Nucleotide Sequencing
Humans
Oligodeoxyribonucleotides
RNA, Guide
RNA Editing

Source:
Nat Biotechnol. 33(2):187-197 ;
Pubmed ID:
25513782
Pubmed Central ID:
PMC4320685
Document Type:
Journal Article ;
Funding:
DP1 GM105378/GM/NIGMS NIH HHS/United States ; DP1 GM105378/DP/NCCDPHP CDC HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States ; F32 GM105189/GM/NIGMS NIH HHS/United States ; R01 AR063070/AR/NIAMS NIH HHS/United States ;
DP1 GM105378/GM/NIGMS NIH HHS/United States ; DP1 GM105378/DP/NCCDPHP CDC HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States ; F32 GM105189/GM/NIGMS NIH HHS/United States ; R01 AR063070/AR/NIAMS NIH HHS/United States ; Less -
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:14fc8c03750b5757e7179488c71ca7db5deba689cf8000b19a91db89aa5b8b1a
File Type:

Supporting Files

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