Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus

Nat Commun. 2017 Jan 9;8:13905. doi: 10.1038/ncomms13905.

Abstract

The CRISPR-Cas9 system provides a versatile toolkit for genome engineering that can introduce various DNA lesions at specific genomic locations. However, a better understanding of the nature of these lesions and the repair pathways engaged is critical to realizing the full potential of this technology. Here we characterize the different lesions arising from each Cas9 variant and the resulting repair pathway engagement. We demonstrate that the presence and polarity of the overhang structure is a critical determinant of double-strand break repair pathway choice. Similarly, single nicks deriving from different Cas9 variants differentially activate repair: D10A but not N863A-induced nicks are repaired by homologous recombination. Finally, we demonstrate that homologous recombination is required for repairing lesions using double-stranded, but not single-stranded DNA as a template. This detailed characterization of repair pathway choice in response to CRISPR-Cas9 enables a more deterministic approach for designing research and therapeutic genome engineering strategies.

MeSH terms

  • BRCA2 Protein / antagonists & inhibitors
  • BRCA2 Protein / genetics*
  • BRCA2 Protein / metabolism
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems*
  • Cell Line, Tumor
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA / genetics*
  • DNA / metabolism
  • DNA Breaks, Double-Stranded
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Gene Editing / methods*
  • Genome, Human*
  • HEK293 Cells
  • Humans
  • K562 Cells
  • Osteoblasts / cytology
  • Osteoblasts / metabolism
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Rad51 Recombinase / antagonists & inhibitors
  • Rad51 Recombinase / genetics*
  • Rad51 Recombinase / metabolism
  • Recombinational DNA Repair*

Substances

  • BRCA2 Protein
  • BRCA2 protein, human
  • Bacterial Proteins
  • RNA, Guide
  • RNA, Small Interfering
  • DNA
  • RAD51 protein, human
  • Rad51 Recombinase
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases