Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair

Nat Commun. 2020 Apr 30;11(1):2109. doi: 10.1038/s41467-020-15845-1.

Abstract

Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away from other repair outcomes. Here, we utilize a pooled CRISPR screen to define host cell involvement in HDR between a Cas9 DSB and a plasmid double stranded donor DNA (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor HDR and that other genes act to repress HDR. Small molecule inhibition of one of these repressors, CDC7, by XL413 and other inhibitors increases the efficiency of HDR by up to 3.5 fold in many contexts, including primary T cells. XL413 stimulates HDR during a reversible slowing of S-phase that is unexplored for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for gene modification.

Publication types

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

MeSH terms

  • CRISPR-Cas Systems*
  • Cell Cycle Proteins / antagonists & inhibitors*
  • Cell Cycle Proteins / genetics*
  • DNA Breaks, Double-Stranded
  • Gene Editing
  • Genetic Engineering / methods
  • HCT116 Cells
  • HEK293 Cells
  • HeLa Cells
  • Homologous Recombination
  • Humans
  • K562 Cells
  • Phenotype
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors*
  • Protein-Serine-Threonine Kinases / genetics*
  • RNA, Guide / metabolism
  • Recombinational DNA Repair*
  • S Phase

Substances

  • Cell Cycle Proteins
  • RNA, Guide
  • CDC7 protein, human
  • Protein-Serine-Threonine Kinases