Journal
YEAST
Volume 31, Issue 3, Pages 103-112Publisher
WILEY-BLACKWELL
DOI: 10.1002/yea.2992
Keywords
MAT alpha 1; mutation; marker-free; seamless; PCR; yeast
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Funding
- National Institutes of Health [5P01HG000205]
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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 MAT alpha 1, with results suggesting that the wild-type gene has a new function outside of that presently known. Copyright (c) 2013 John Wiley & Sons, Ltd.
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