Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant

Nat Commun. 2019 Jul 30;10(1):3395. doi: 10.1038/s41467-019-11105-z.

Abstract

Precise editing of genomic DNA can be achieved upon repair of CRISPR-induced DNA double-stranded breaks (DSBs) by homology-directed repair (HDR). However, the efficiency of this process is limited by DSB repair pathways competing with HDR, such as non-homologous end joining (NHEJ). Here we individually express in human cells 204 open reading frames involved in the DNA damage response (DDR) and determine their impact on CRISPR-mediated HDR. From these studies, we identify RAD18 as a stimulator of CRISPR-mediated HDR. By defining the RAD18 domains required to promote HDR, we derive an enhanced RAD18 variant (e18) that stimulates CRISPR-mediated HDR in multiple human cell types, including embryonic stem cells. Mechanistically, e18 induces HDR by suppressing the localization of the NHEJ-promoting factor 53BP1 to DSBs. Altogether, this study identifies e18 as an enhancer of CRISPR-mediated HDR and highlights the promise of engineering DDR factors to augment the efficiency of precision genome editing.

Publication types

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

MeSH terms

  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA Damage
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Gene Editing
  • Humans
  • Protein Domains
  • Protein Engineering
  • Recombinational DNA Repair
  • Ubiquitin-Protein Ligases / chemistry
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*

Substances

  • DNA-Binding Proteins
  • RAD18 protein, human
  • Ubiquitin-Protein Ligases