Development of a Donor Vector for Efficient CRISPR/Cas9-Mediated Homology-Directed Integration at the CAN1 Locus in...

Saccharomyces cerevisiae is a widely utilized host organism for recombinant protein production due to its robustness, versatility, and ease of genetic manipulation. Gene integration into its genome is a critical strategy for achieving stable and efficient protein expression. However, incorporating large gene cassettes at specific loci, such as the CAN1 locus, remains technically challenging. While CRISPR/Cas9-based genome editing has significantly improved precision, the efficiency of integrating large constructs can still be limited. In this study, we optimized a CRISPR/Cas9-based approach using the pBlueHA donor vector, which contains approximately 500 bp homology arms flanking the CAN1 locus, to enhance gene integration efficiency. By testing 13 genes of interest (GOIs) of various sizes, up to 4756 bp in size, we successfully achieved integration into the yeast genome. Linearized GOIs ranging exhibited significantly higher integration efficiencies, ranging from 85.3 to 100%, compared to circular cassettes. These results highlight the importance of extended homology arms in improving integration efficiency, providing a robust platform for optimizing genome engineering in S. cerevisiae to enhance recombinant protein production and other biotechnological applications.

The online version contains supplementary material available at 10.1007/s12088-025-01457-0.