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

Holly A ReesWei-Hsi YehDavid R Liu
Published in: Nature communications (2019)
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.
Keyphrases
  • crispr cas
  • genome editing
  • single cell
  • circulating tumor
  • copy number
  • cell therapy
  • cell free
  • gene expression
  • dna methylation
  • genome wide