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

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalNot applicablepeer-review

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Author(s)

  • Shengdar Q Tsai
  • Nhu T Nguyen
  • Matthew Liebers
  • Ved V Topkar
  • Vishal Thapar
  • Nicolas Wyvekens
  • Cyd Khayter
  • A John Iafrate
  • Long P Le
  • Martin J Aryee
  • J Keith Joung

Detail(s)

Original languageEnglish
Pages (from-to)187-198
Journal / PublicationNature Biotechnology
Volume33
Issue number2
Early online date16 Dec 2014
Publication statusPublished - Feb 2015
Externally publishedYes

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.

Citation Format(s)

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

In: Nature Biotechnology, Vol. 33, No. 2, 02.2015, p. 187-198.

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalNot applicablepeer-review