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

Abstract

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. 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. 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.