The CRISPR-Cas9 system from Streptococcus pyogenes has revolutionized genome modification through precise editing across a wide range of organisms. Yeast supports efficient genome editing via plasmid-based Cas9-gRNA expression, while higher eukaryotes often require genome-integrated cassettes or RNP delivery. In this study, we engineered CRISPR components to enhance nuclear targeting and editing efficiency. We demonstrated the proof of concept in Saccharomyces cerevisiae using its CAN1 locus. We developed a dual-host compatible vector, encoding Cas9 nuclease fused with three nuclear localization signals (Cas9-3xNLS). The recombinant protein, expressed in E. coli and purified on a Ni-NTA column, showed DNA cleavage in an in vitro assay. Genome editing efficacy of Cas9-3xNLS was demonstrated in S. cerevisiae AH109 strain. Further, we engineered sgRNAs by extending their ends to facilitate the annealing to ssODN. We synthesized ssODNs having a complementary sequence either at 3' or 5' to anchor with sgRNAs. sgRNAs (unmodified and end extended) and ssODNs were introduced into yeast in various combinations. sgRNA with a 3' ssDNA-anchoring motif and ssODN representing the antisense strand of the target gene with sgRNA complementary motif at the 5' end (free homology arm at 3') improved HDR efficiency significantly. This combination yielded about a 1.64-fold increase in canavanine-resistant colonies as compared to the control via precise insertion of a stop codon. In contrast, extension of sgRNA at the 5' end did not show any advantage. This approach is flexible and easy to use and has the potential to enhance homology-directed repair in diverse organisms.
