CRISPR/Cas9‐Based Genome Editing of the Saccharomyces cerevisiae ADE2 Gene with Restriction‐Free Cloning and a Rapid BamHI Digest Readout

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‐associated systems (CRISPR/Cas) are revolutionary tools for predictable and repeatable genome editing. In this article, the CRISPR/Cas9 system will be used to engineer the genome of the yeast Saccharomyces cerevisiae. As a model organism utilized across biological disciplines, efficient and tailorable methods for engineering the yeast genome are indispensable for a variety of research, educational, and commercial applications. The protocols described here incorporate a simple restriction‐free cloning strategy and digestion‐based screening method that can be readily and easily modified to suit user‐designed experimental needs. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Recombinant pCAS plasmid preparation Basic Protocol 2: Barcode/editing fragment assembly Basic Protocol 3: Gene editing by yeast co‐transformation Alternate Protocol: Competent yeast preparation and transformation by a lithium acetate/single‐stranded carrier method