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

Author

Listed:
  • Holly A. Rees

    (Broad Institute of Harvard and MIT
    Harvard University
    Harvard University)

  • Wei-Hsi Yeh

    (Broad Institute of Harvard and MIT
    Harvard University
    Harvard University
    Harvard Medical School)

  • David R. Liu

    (Broad Institute of Harvard and MIT
    Harvard University
    Harvard University)

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.

Suggested Citation

  • Holly A. Rees & Wei-Hsi Yeh & David R. Liu, 2019. "Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks," Nature Communications, Nature, vol. 10(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-09983-4
    DOI: 10.1038/s41467-019-09983-4
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    Cited by:

    1. Lukas Möller & Eric J. Aird & Markus S. Schröder & Lena Kobel & Lucas Kissling & Lilly van de Venn & Jacob E. Corn, 2022. "Recursive Editing improves homology-directed repair through retargeting of undesired outcomes," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    2. Zsolt Bodai & Alena L. Bishop & Valentino M. Gantz & Alexis C. Komor, 2022. "Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies," Nature Communications, Nature, vol. 13(1), pages 1-15, December.
    3. J. Ferreira da Silva & G. P. Oliveira & E. A. Arasa-Verge & C. Kagiou & A. Moretton & G. Timelthaler & J. Jiricny & J. I. Loizou, 2022. "Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    4. Michael Kosicki & Felicity Allen & Frances Steward & Kärt Tomberg & Yangyang Pan & Allan Bradley, 2022. "Cas9-induced large deletions and small indels are controlled in a convergent fashion," Nature Communications, Nature, vol. 13(1), pages 1-11, December.

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