Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52

Int J Biochem Cell Biol. 2017 Nov;92:43-52. doi: 10.1016/j.biocel.2017.09.012. Epub 2017 Sep 18.

Abstract

Precise genome editing with desired point mutations can be generated by CRISPR/Cas9-mediated homology-directed repair (HDR) and is of great significance for gene function study, gene therapy and animal breeding. However, HDR efficiency is inherently low and improvements are necessitated. Herein, we determined that the HDR efficiency could be enhanced by expressing Rad52, a gene that is involved in the homologous recombination process. Both the Rad52 co-expression and Rad52-Cas9 fusion strategies yielded approximately 3-fold increase in HDR during the surrogate reporter assays in human HEK293T cells, as well as in the genome editing assays. Moreover, the enhancement effects of the Rad52-Cas9 fusion on HDR mediated by different (plasmid, PCR and ssDNA) donor templates were confirmed. We found that the HDR efficiency could be significantly improved to about 40% by the combined usage of Rad52 and Scr7. In addition, we also applied the fusion strategy for modifying the IGF2 gene of porcine PK15 cells, which further demonstrated a 2.2-fold increase in HDR frequency. In conclusion, our data suggests that Rad52-Cas9 fusion is a good option for enhancing CRISPR/Cas9-mediated HDR, which may be of use in future studies involving precise genome editing.

Keywords: CRISPR/Cas9; Homology-directed repair; Precise genome editing; Rad52; Surrogate reporter.

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • DNA Breaks, Double-Stranded
  • DNA Repair / genetics*
  • Gene Editing / methods*
  • Gene Expression
  • Genomics
  • HEK293 Cells
  • Homologous Recombination
  • Humans
  • Insulin-Like Growth Factor II / genetics
  • Point Mutation
  • Rad52 DNA Repair and Recombination Protein / genetics*
  • Saccharomyces cerevisiae / genetics*
  • Sequence Homology, Nucleic Acid*

Substances

  • IGF2 protein, human
  • RAD52 protein, human
  • Rad52 DNA Repair and Recombination Protein
  • Insulin-Like Growth Factor II