The 50:50 Method for PCR-based Seamless Genome Editing in Yeast

摘要

The ability to edit the yeast genome with relative ease has contributed to the organism being a model eukaryote for decades. Most methods for deleting, inserting, or altering genomic sequences require transformation with DNA that carries the desired change and a selectable marker. One-step genome editing methods retain the selectable marker. Seamless genome editing methods require more steps and a marker that can be used for both positive and negative selection, such as URA3. Here we describe the PCR-based Fifty-Fifty method for seamless genome editing that requires only two primers, one PCR with a URA3 cassette, and a single yeast transformation. Our method is based on pop-in/pop-out gene replacement and is amenable to the facile creation of genomic deletions and short insertions or substitutions. We used the Fifty-Fifty method to make two conservative loss-of-function mutations in MATALPHA1, with results that suggest the wildtype gene has a new function outside of that presently known.