The 50:50 method for PCR‐based seamless genome editing in yeast

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 50:50 method for seamless genome editing, which 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 50:50 method to make two conservative loss‐of‐function mutations in MATα1 , with results suggesting that the wild‐type gene has a new function outside of that presently known. Copyright © 2013 John Wiley & Sons, Ltd.