Protocol for CRISPR genome editing in S. cerevisiae using PCR-based guide insertion

Here, we present a CRISPR-Cas9 genome-editing protocol for Saccharomyces cerevisiae that simplifies guide construction and enables rapid guide iteration by replacing restriction/ligation cloning with PCR-based guide installation and seamless Cas9-plasmid recircularization. We describe single-guide RNA (sgRNA) and homology-directed repair (HDR) donor design, guide insertion into a KanMX/G418-selectable Cas9-sgRNA plasmid, plasmid cloning and sequence verification in E. coli, and lithium acetate/polyethylene glycol (LiAc/PEG) co-transformation of yeast with the verified plasmid and HDR donor.