Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks
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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- May 17, 2019
- Source ID
- 10.1038/s41467-019-09983-4
Entities
People
- David R. Liu
- Holly A. Rees
- Wei-Hsi Yeh
Organizations
- National Human Genome Research Institute
- National Institute of Allergy and Infectious Diseases
- National Institute of Biomedical Imaging and Bioengineering
- National Institute of General Medical Sciences
- United States Department of Defense