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

Holly A. Rees, Wei-Hsi Yeh and David R. Liu ()
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Holly A. Rees: Broad Institute of Harvard and MIT
Wei-Hsi Yeh: Broad Institute of Harvard and MIT
David R. Liu: Broad Institute of Harvard and MIT

Nature Communications, 2019, vol. 10, issue 1, 1-12

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-09983-4

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