title: High-efficiency genome editing and allele replacement in prototrophic and wild strains of Saccharomyces.
abstract: Current genome editing techniques available for Saccharomyces yeast species rely on auxotrophic markers, limiting their use in wild and industrial strains and species. Taking advantage of the ancient loss of thymidine kinase in the fungal kingdom, we have developed the herpes simplex virus thymidine kinase gene as a selectable and counterselectable marker that forms the core of novel genome engineering tools called the H: aploid E: ngineering and R: eplacement P: rotocol (HERP) cassettes. Here we show that these cassettes allow a researcher to rapidly generate heterogeneous populations of cells with thousands of independent chromosomal allele replacements using mixed PCR products. We further show that the high efficiency of this approach enables the simultaneous replacement of both alleles in diploid cells. Using these new techniques, many of the most powerful yeast genetic manipulation strategies are now available in wild, industrial, and other prototrophic strains from across the diverse Saccharomyces genus.
authors.affiliation: Laboratory of Genetics, Genome Center of Wisconsin, Wisconsin Energy Institute, J. F. Crow Institute for the Study of Evolution, University of Wisconsin, Madison, Wisconsin 53706 DOE Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: William G
authors.initials: WG
authors.lastname: Alexander
authors.affiliation: Laboratory of Genetics, Genome Center of Wisconsin, Wisconsin Energy Institute, J. F. Crow Institute for the Study of Evolution, University of Wisconsin, Madison, Wisconsin 53706 Graduate Program in Cellular and Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Drew T
authors.initials: DT
authors.lastname: Doering
authors.affiliation: Laboratory of Genetics, Genome Center of Wisconsin, Wisconsin Energy Institute, J. F. Crow Institute for the Study of Evolution, University of Wisconsin, Madison, Wisconsin 53706 DOE Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, Wisconsin 53706 Graduate Program in Cellular and Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706 cthittinger@wisc.edu.
authors.forename: Chris Todd
authors.initials: CT
authors.lastname: Hittinger
mesh_terms: D000483:Alleles; D002454:Cell Differentiation; D015825:Chromosomes, Fungal / Q000235:genetics; D004171:Diploidy; D005819:Genetic Markers; D016681:Genome, Fungal / Q000235:genetics*; D006720:Homozygote; D016254:Mutagenesis, Insertional; D017393:RNA Editing / Q000235:genetics*; D012440:Saccharomyces / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D013937:Thymidine Kinase / Q000378:metabolism
chemical_list: D005819:Genetic Markers; D013937:Thymidine Kinase
keywords: Saccharomyces; genome editing; prototroph; synthetic biology; thymidine kinase