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 Haploid Engineering and Replacement Protocol (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.G ENOME editing is a precise and powerful tool to investigate basic genetic processes or to reprogram an organism's metabolism. Techniques to precisely manipulate genomes exist for many model organisms (Storici et al. 2003;Gratz et al. 2013;Hwang et al. 2013;Jiang et al. 2013;Tzur et al. 2013), but not all features of these approaches are easily portable to closely related species. The genus Saccharomyces is highly experimentally tractable, and laboratory strains of all seven natural species can be genetically manipulated Liti et al. 2013). Saccharomyces cerevisiae is the most well-known member of the genus due to its role in brewing (Lodolo et al. 2008), biofuels (Steen et al. 2008;Matsushika et al. 2009), winemaking (Peris et al. 2012, and baking, as well as a model system for the biological sciences (Botstein and Fink 2011). Other members of the genus are also used by humans in the form of interspecies hybrids, such as the S. cerevisiae 3 Saccharomyces kudriavzevii hybrids used to ferment some wines and Belgian beers (Peris et al. 2012) and the S. cerevisiae 3 Saccharomyces eubayanus (Libkind et al. 2011) hybrids found in the brewing of lager-style beers around the world. The Saccharomyces genus is also an emerging "model genus" for molecular evolution, and several experimentally tractable species are now used routinely in evolutionary genetics research (Hittinger 2013). Efficient genome editing of these diverse Saccharomyces yeasts would therefore provide new avenues of investigation for basic and applied research.One major reason for the popularity of S. cerevisiae as a model system is the availability of powerful genetic manipulation tools. One of these tools is the URA3 selection/counterselection system (Boeke et al. 1984). URA3 is a gene required for the de novo synthesis of uracil. Thus, the URA3 gene can be used as a selectable marker in ura3 strains by selecting for the ability to grow on synthetic media without uracil. The deactivation or replacement of URA3 can also be selected for using synt...

Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production

“…Heterothallic haploid derivatives of Y73 (GLBRCY101) and S. eubayanus FM1318 (yHEB67) were generated in this study. For the generation of haploid derivative strains, the HO locus was replaced by a drug-resistance or TK [88] marker, and transformants were selected (Table 1). Sporulation was induced on sporulation plates (1% Potassium Acetate, 0.1% yeast extract, 0.05% glucose, 2% agar) or YPD at 24 °C, and tetrads were microdissected and micromanipulated using the SporePlay Dissector (Singer Instruments, UK).…”

Efficient engineering of marker-free synthetic allotetraploids of Saccharomyces

“…As with a previous case where HO was induced in a sterile strain whose genome was predominantly S. uvarum (Schwartz et al, 2012), HyPr is expected to even enable hybridization of strains whose defect is in sporulation or chromosome segregation, rather than mating per se . This new chassis strain could then undergo optimization via selective conditions, further enhancing desired phenotypes of the chassis strain through aneuploidy and other mutations or through modification using various genome-editing techniques (Alexander et al, 2014; DiCarlo et al, 2013; Ryan et al, 2014). …”

Ongoing resolution of duplicate gene functions shapes the diversification of a metabolic network

“…Transformations were based on the standard lithium acetate/PEG method optimized for S. uvarum (room temperature incubation, followed by a 37˚C heat shock) (Gietz et al, 1995; Caudy et al, 2013). To perform allele swaps, the coding sequence or promoter was first replaced by a selectable and counter-selectable TK-hphMX cassette, which does not require the prior introduction of an auxotrophy (Alexander et al, 2014). The coding sequence or promoter of the desired replacement sequence was amplified by PCR primers with overhangs homologous to the targeted genomic flanking region.…”

“…Note that the S. cerevisiae and S. uvarum GAL1 promoters are both divergent promoters that also regulate GAL10 and may also impact a lncRNA previously described in S. cerevisiae (Cloutier et al, 2016). Successful replacement strains were isolated by selecting for the loss of thymidine kinase activity by resistance to 5-fluorodeoxyuridine (FUdR), as well as the loss of resistance to hygromycin by replica plating (Alexander et al, 2014). GFP reporters were constructed in three parts: the hphMX cassette was placed upstream as the selection marker, the S. uvarum GAL1 promoter was used to drive the expression of the reporter, and the reporter was a yEGFP (yeast Enhanced Green Fluorescence Protein) construct with a S. cerevisiae CYC1 terminator that was amplified from FM1282 (Hittinger and Carroll, 2007; Hittinger et al, 2010).…”