Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

Science. 2019 Apr 19;364(6437):286-289. doi: 10.1126/science.aav9023. Epub 2019 Apr 18.

Abstract

CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.

Publication types

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

MeSH terms

  • Adenoviridae
  • Animals
  • CRISPR-Associated Protein 9 / chemistry
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Cell Line
  • Chromatin Immunoprecipitation
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA / chemistry
  • DNA / genetics
  • DNA Breaks, Double-Stranded*
  • DNA Repair Enzymes / metabolism
  • DNA Repair*
  • Gene Editing / methods*
  • Humans
  • Induced Pluripotent Stem Cells
  • K562 Cells
  • MRE11 Homologue Protein / genetics
  • MRE11 Homologue Protein / metabolism*
  • RNA, Guide
  • Sequence Analysis, DNA / methods*

Substances

  • MRE11 protein, human
  • RNA, Guide
  • DNA
  • CRISPR-Associated Protein 9
  • MRE11 Homologue Protein
  • DNA Repair Enzymes