Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor

Despite the potential of small molecules and recombinant proteins to improve homology-directed repair (HDR) efficiency, single-stranded DNA (ssDNA) donors, in their current designs and chemical modifications, remain suboptimal for precise gene editing. In this study, we screen ssDNA-binding sequences favored by DNA repair-related proteins and incorporate RAD51-preferred sequences into specialized HDR-enhancing modules for ssDNA donors. These modified donors show increased affinity for RAD51, significantly boosting M3814 HDR efficiency across various genomic sites and cell types when paired with Cas9, nCas9, or Cas12a. When combined with a non-homologous end joining (NHEJ) inhibitor or the HDRobust strategy, these modular ssDNA donors achieve HDR efficiencies as high as 90.03% (median 74.81%). By targeting an endogenous protein, the HDR-enhancing modules provide a chemical modification-free solution to improve the effectiveness of ssDNA donors for precise gene editing.