Prime Editing-Mediated Genome Modification in Saccharomyces cerevisiae.

Prime editing is a highly versatile genome editing technology that enables precise genetic modifications in eukaryotes. Here, we present a detailed step-by-step protocol for conducting prime editing in Saccharomyces cerevisiae. In particular, we describe the design of prime editing guide RNAs (pegRNAs), yeast transformation with prime editor and pegRNA plasmids, and controlled expression through galactose induction. We demonstrate the protocol by targeting the CAN1 gene and achieving editing efficiencies of 22% and 55% with two different pegRNAs. We provide protocols for genomic DNA extraction, PCR amplification, and editing quantification using both Sanger and next-generation sequencing. Given the important role of yeast in both basic research and biotechnology applications, this protocol provides valuable tools for studying gene function and precise engineering of yeast strains.