Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR-Cas9 genome editing

Nat Biomed Eng. 2017 Nov;1(11):878-888. doi: 10.1038/s41551-017-0145-2. Epub 2017 Oct 9.

Abstract

Gene disruption by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is highly efficient and relies on the error-prone non-homologous end-joining pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than non-homologous end-joining in mammalian cells. Here, by testing whether manipulation of DNA repair factors improves HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative form of tumour protein p53-binding protein 1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of non-homologous end-joining-mediated double-strand break repair in the presence of these two factors is not suppressed and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.

Publication types

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

MeSH terms

  • CRISPR-Cas Systems*
  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair
  • DNA Repair*
  • Ectopic Gene Expression
  • Gene Editing / methods*
  • HEK293 Cells
  • Humans
  • Induced Pluripotent Stem Cells / metabolism
  • Rad52 DNA Repair and Recombination Protein / genetics*
  • Recombinational DNA Repair
  • Tumor Suppressor p53-Binding Protein 1 / genetics*

Substances

  • RAD52 protein, human
  • Rad52 DNA Repair and Recombination Protein
  • TP53BP1 protein, human
  • Tumor Suppressor p53-Binding Protein 1