Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens

Nat Commun. 2017 May 5;8:15178. doi: 10.1038/ncomms15178.

Abstract

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.

Publication types

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

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • DNA Damage / genetics
  • Gene Targeting / methods*
  • Genomic Library*
  • High-Throughput Screening Assays / methods*
  • Humans
  • Polysaccharides / biosynthesis
  • RNA Interference
  • RNA, Guide / genetics*
  • Ricin / toxicity

Substances

  • Polysaccharides
  • RNA, Guide
  • Ricin