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

Nat Biotechnol. 2015 Feb;33(2):187-197. doi: 10.1038/nbt.3117. Epub 2014 Dec 16.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • DNA Breaks, Double-Stranded*
  • Genome, Human
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Oligodeoxyribonucleotides / genetics
  • RNA Editing / genetics
  • RNA, Guide / genetics*

Substances

  • Oligodeoxyribonucleotides
  • RNA, Guide