Step-by-step method for editing yeast genes using CRISPR with PCR-made guides

A CRISPR-Cas9 genome-editing protocol for Saccharomyces cerevisiae eliminates the need for restriction/ligation cloning.

• The protocol simplifies guide construction by using PCR-based methods.
• Rapid guide iteration is enabled through seamless Cas9-plasmid recircularization.
• Design processes for single-guide RNA (sgRNA) and homology-directed repair (HDR) donors are outlined.
• The method includes guide insertion into a selectable plasmid for yeast transformation.
• Sequence verification of the plasmid is performed in E. coli prior to yeast transformation.

Simplified