Enhancement of single-stranded template annealing activity by Rad52 during repair of CRISPR-induced dsDNA breaks - PubMed
4 hours ago
- #CRISPR Genome Editing
- #Rad52
- #Single-Strand Annealing
- Single-strand annealing protein (SSAP)-mediated recombination engineering is powerful for bacterial genome editing but limited in eukaryotes by dominant NHEJ and low exogenous SSAP activity.
- In NHEJ-dominated Yarrowia lipolytica, 18.7% of Cas9-induced DSBs were precisely repaired with single-stranded oligonucleotide templates without recombinase overexpression, indicating endogenous eukaryotic SSAP activity.
- Rad52 was identified as key in single-strand annealing; its structural truncation (Rad52(1-300)) boosted genome-editing efficiency to 96.3%, comparable to disrupting NHEJ via Ku70 deletion.
- The ESTAR platform leverages Rad52 to enable precise, efficient genome editing, including small-fragment insertions/deletions/replacements and large-fragment deletions over 20 kb.
- This editing technology was validated in Saccharomyces cerevisiae and other non-conventional yeasts, offering new insights into single-stranded DNA annealing during Cas9-induced DSB repair.