Development of hRad51-Cas9 nickase fusions that mediate HDR without double-stranded breaks

Nat Commun. 2019 May 17;10(1):2212. doi: 10.1038/s41467-019-09983-4.

Abstract

In mammalian cells, double-stranded DNA breaks (DSBs) are preferentially repaired through end-joining processes that generally lead to mixtures of insertions and deletions (indels) or other rearrangements at the cleavage site. In the presence of homologous DNA, homology-directed repair (HDR) can generate specific mutations, albeit typically with modest efficiency and a low ratio of HDR products:indels. Here, we develop hRad51 mutants fused to Cas9(D10A) nickase (RDN) that mediate HDR while minimizing indels. We use RDN to install disease-associated point mutations in HEK293T cells with comparable or better efficiency than Cas9 nuclease and a 2.7-to-53-fold higher ratio of desired HDR product:undesired byproducts. Across five different human cell types, RDN variants generally result in higher HDR:indel ratios and lower off-target activity than Cas9 nuclease, although HDR efficiencies remain strongly site- and cell type-dependent. RDN variants provide precision editing options in cell types amenable to HDR, especially when byproducts of DSBs must be minimized.

Publication types

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

MeSH terms

  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism*
  • DNA Breaks, Double-Stranded
  • Gene Editing / methods
  • Genetic Engineering / methods*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Induced Pluripotent Stem Cells
  • K562 Cells
  • Rad51 Recombinase / genetics
  • Rad51 Recombinase / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Recombinational DNA Repair*
  • Transfection / methods

Substances

  • Recombinant Fusion Proteins
  • RAD51 protein, human
  • Rad51 Recombinase
  • CRISPR-Associated Protein 9