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April 1st, 2025
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University of Kent
genomics
biorxiv

CRISPR/Cas9-mediated editing of the ADE2 locus in Saccharomyces cerevisiae: targeted mutagenesis and homology-directed repair

JARRA, E.Open in Google Scholar

The CRISPR/Cas9 system, derived from the adaptive immune defence of bacteria and archaea, has emerged as a powerful tool for genome engineering by enabling precise, site-specific modifications. Type II bacterial CRISPR systems recognize and cleave DNA in various microorganisms using the RNA-guided Cas9 endonuclease. We present a streamlined and efficient approach for genome editing in two strains of Saccharomyces cerevisiae, focused on targeted mutagenesis of the ADE2 gene of the BY4741 strain and homology-directed repair of the ade2 locus of the W303-1A strain, involving a single-step transformation strategy utilizing the pML104 plasmid, which encodes the Cas9 endonuclease and a user-defined single-guide RNA (gRNA) complementary to the target site. In the BY4741 (wildtype) strain, pML104-gRNA components achieved successful ADE2 disruption rates of 40% (95% CI: 0.10--0.70), confirmed by adenine auxotrophy screening and Sanger sequencing. Additionally, co-transformation of gRNA plasmid and donor repair templates in the W303-1A (ade2 mutant) strain also resulted in 40% recombination efficiency, restoring the function of the mutant gene to allow activation of de novo purine biosynthesis activity when cultured on SC-Ade medium. This optimized, cost-effective CRISPR/Cas9 method enhances editing efficiency in S. cerevisiae, providing a robust and practical platform for high-precision genome modifications with broad applications in molecular biology and biotechnology.

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