Efficient targeted gene knock-in in Ulva using Cas9 RNPs and long single-stranded DNA donors

Abstract

Abstract Background CRISPR-Cas9 ribonucleoprotein (RNP)-mediated genome editing has recently been established in the green seaweed Ulva . However, achieving precise and efficient targeted gene insertion remains challenging due to the low frequency of homology-directed repair (HDR) and suboptimal donor DNA design. In this study, we attempted to optimize a knock-in strategy by co-delivering Cas9 RNPs and donor DNA templates to target the highly expressed RbcS gene for EGFP insertion, while simultaneously disrupting the adenine phosphoribosyltransferase ( APT ) gene for robust selection. We compared the efficacy of single-stranded (ssDNA) versus double-stranded (dsDNA) donors with varying homology arm (HA) lengths. Results We found that ssDNA donors significantly outperformed dsDNA templates. Furthermore, 50-nt HAs were ineffective, while ssDNA donors with 300-nt HAs achieved the highest insertion efficiency. Sequence analysis revealed the loss of a donor-specific deletion, suggesting that Ulva utilizes synthesis-dependent strand annealing (SDSA) or mismatch repair pathways, rather than the microhomology-mediated mechanisms prevalent in Chlamydomonas . The APT -based co-targeting strategy effectively enriched the candidate population, enabling a discovery rate of approximately 3% for EGFP-positive strains among resistant individuals, achieving the first successful generation of a targeted double mutant in this species. Additionally, using tandem 2 A peptides (P2A-T2A) significantly improved ribosomal skipping efficiency compared to single 2 A systems, facilitating effective polycistronic expression. Conclusions Collectively, this study establishes a streamlined and highly efficient framework for precise insertional mutagenesis and double-mutant generation in Ulva , thereby expanding the genetic engineering toolkit for this macroalga.