Yeast Genetic Manipulation and Cas9 Integration

Saccharomyces cerevisiae strains were transformed according to Gietz and Woods (2002 ). Mutants were selected on solid YP medium (demineralized water, 10 g·L⁻¹ Bacto yeast extract, 20 g·L⁻¹ Bacto peptone, 2% (w/v) agar), supplemented with 200 mg·L⁻¹ G418, 200 mg·L⁻¹ hygromycin B or 100 mg·L⁻¹ nourseothricin (for dominant markers) or on SM supplemented with appropriate auxotrophic requirements (Verduyn et al.1992 (link)). In all cases, gene deletions and integrations were confirmed by colony PCR on randomly picked colonies, using the diagnostic primers listed in Table S1 (Supplementary data). Integration of cas9 into the genome was achieved via assembly and integration of two cassettes containing cas9 and the natNT2 marker into the CAN1 locus. The cas9 cassette was obtained by PCR from p414-TEF1p-cas9-CYC1t (DiCarlo et al.2013b (link)), using primers 2873 & 4653. The natNT2 cassette was PCR amplified from pUG-natNT2 with primers 3093 & 5542. 2.5 μg cas9 and 800 ng natNT2 cassette were pooled and used for each transformation. Correct integration was verified by colony PCR (Supplementary data) using the primers given in Table S1 (Supplementary data), the resulting strains have been deposited at EUROSCARF. IMX719 was constructed by co-transformation of pUDR022 (see below) with genes required for functional Enterococcus faecalis PDH expression (Kozak et al.2014b (link)). The gene cassettes were obtained by PCR using plasmids pUD301–pUD306 as template (Table 2) with the primers indicated in Table S1 (Supplementary data) and the ACS1 dsDNA repair fragment, obtained by annealing two complementary single-stranded oligos (6422 & 6423). After confirmation of the relevant genotype (Fig. 4B), the pUDR022 plasmid was removed as explained in Supplementary data.