Genome-scale CRISPR screens are efficient in non-homologous end-joining deficient cells

Sci Rep. 2019 Oct 31;9(1):15751. doi: 10.1038/s41598-019-52078-9.

Abstract

The mutagenic repair of Cas9 generated breaks is thought to predominantly rely on non-homologous end-joining (NHEJ), leading to insertions and deletions within DNA that culminate in gene knock-out (KO). In this study, by taking focused as well as genome-wide approaches, we show that this pathway is dispensable for the repair of such lesions. Genetic ablation of NHEJ is fully compensated for by alternative end joining (alt-EJ), in a POLQ-dependent manner, resulting in a distinct repair signature with larger deletions that may be exploited for large-scale genome editing. Moreover, we show that cells deficient for both NHEJ and alt-EJ were still able to repair CRISPR-mediated DNA double-strand breaks, highlighting how little is yet known about the mechanisms of CRISPR-based genome editing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair
  • Gene Editing / methods*
  • Gene Knockout Techniques
  • HSP90 Heat-Shock Proteins / genetics
  • Humans
  • RNA, Guide / metabolism
  • Ubiquitin-Protein Ligases / genetics

Substances

  • HSP90 Heat-Shock Proteins
  • HSP90AA1 protein, human
  • RNA, Guide
  • RNF152 protein, human
  • Ubiquitin-Protein Ligases
  • CRISPR-Associated Protein 9