Fig. 1.

Evidence for rational design of odsDNA donor for gene-sized KI. (A) A known putative model for precise Cas9-induced DSB repair using ssDNA donor. The 3′-overhangs flanking the DSB breakage site in the genome are exposed following bidirectional end resection (i). The 3′ ssDNA end anneals to one DSB end via microhomology-mediated SSA and is thus efficiently repaired through the non-canonical HDR-mediated SSA pathway (ii). At the 5′ ssDNA donor end, owing to the lack of complementarity between 5′ ssDNA end and the DSB overhang, it is precisely repaired through relatively low-efficient HDR dependent on synthesis-dependent strand annealing (SDSA) (iii). (B) HDR-mediated repair for Cas9-generated DSBs using dsDNA donor. Both the newly produced DSB overhangs independently invade the dsDNA donor template and synthesize new strand with the dsDNA template. Both DSB ends are precisely repaired through HR-mediated HDR. (C) A rational design for a hybrid ‘ssDNA-dsDNA’ donor model, referred to as 3′-overhang dsDNA (odsDNA). (D) In this hypothesized odsDNA model, both 3′- overhangs of odsDNA would bias the DSB repair toward more efficient SSA-mediated non-canonical HDR repair, similar to the 3′ ssDNA end repair in A. Overhang binding site, OBS. The structure of Cas9 (no. 4CMP) from PDB database.