Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 39122671.
- Also identified by DOI 10.1038/s41467-024-50788-x and PMC identifier 11315919.
- Licence recorded as CC BY-NC-ND.
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Abstract
Despite the potential of small molecules and recombinant proteins to enhance the efficiency of homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed and chemically modified, remain suboptimal for precise gene editing. Here, we screen the biased ssDNA binding sequences of DNA repair-related proteins and engineer RAD51-preferred sequences into HDR-boosting modules for ssDNA donors. Donors with these modules exhibit an augmented affinity for RAD51, thereby enhancing HDR efficiency across various genomic loci and cell types when cooperated with Cas9, nCas9, and Cas12a. By combining with an inhibitor of non-homologous end joining (NHEJ) or the HDRobust strategy, these modular ssDNA donors achieve up to 90.03% (median 74.81%) HDR efficiency. The HDR-boosting modules targeting an endogenous protein enable a chemical modification-free strategy to improve the efficacy of ssDNA donors for precise gene editing.
Medical subject headings
- DNA, Single-Stranded
- Gene Editing
- Recombinational DNA Repair
- Rad51 Recombinase