Enhancement of single-stranded template annealing activity by Rad52 during repair of CRISPR-induced dsDNA breaks

Single-strand annealing protein (SSAP)-mediated recombination engineering has become a powerful tool for bacterial genome editing. However, in most eukaryotes, its efficiency is constrained by the dominant non-homologous end joining (NHEJ) repair pathway and the limited activity of exogenous SSAPs. Here, in the typically NHEJ-dominated yeast Yarrowia lipolytica, we found that 18.7% of Cas9-induced double-strand breaks (DSBs) were precisely repaired upon provision of single-stranded oligonucleotide templates, even in the absence of recombinase overexpression, indicating the presence of an endogenous eukaryotic SSAP-mediated recombination activity. Overexpression of recombination-related proteins revealed that Rad52 plays a key role in single-strand annealing. Structural truncation of Rad52(1-300) boosted genome-editing efficiency to 96.3%, comparable to that achieved by disrupting NHEJ via Ku70 deletion. Our ESTAR platform (enhancement of single-stranded template annealing activity by Rad52) enables precise and efficient genome editing, including small-fragment insertions, deletions, and replacements, as well as large-fragment deletions exceeding 20 kb. This gene-editing technology was further validated in Saccharomyces cerevisiae and other non-conventional yeast, offering new insights into the single-stranded DNA annealing step during the repair of Cas9-induced DSBs.