title: The 50:50 method for PCR-based seamless genome editing in yeast.
abstract: 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.
authors.affiliation: Department of Biochemistry, Stanford University, CA, USA.
authors.forename: Joe
authors.initials: J
authors.lastname: Horecka
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
mesh_terms: D017931:DNA Primers / Q000235:genetics; D017353:Gene Deletion; D018390:Gene Targeting; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics; D016681:Genome, Fungal / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D005819:Genetic Markers; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C554167:URA3 protein, S cerevisiae
keywords: MATα1; PCR; marker‐free; mutation; seamless; yeast