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To knock out genes in S. cerevisiae, we used a Cas9-assisted homologous recombination approach:
- Build an S. cerevisiae shuttle vector expressing Cas9 and a guide RNA targeting the gene of interest.
- Use PCR to make a linear URA3 cassette flanked by homologous sequences upstream and downstream of the Cas9 target site.
- Transform BY4735 with the plasmid and PCR DNA. Select on Ura dropout plates.
- Use PCR to verify the URA3 insertion.
- Background strain: BY4735. BY4735 is a MATalpha 6-way auxotroph based on S288C, bearing non-revertable deletions in ADE2, HIS3, LEU2, MET15, TRP1 and URA3.
- Plasmids: Knock-out plasmids were built using the Dueber lab's yeast toolkit. Additional assembly details are below.
Construction of Cas9/sgRNA plasmids
- We used the Yeast Toolkit to build a L2 plasmid containing the following functional parts:
- Cas9 driven by the constitutive PGK1 promoter
- an sgRNA cassette with a GFP dropout
- a LEU2 yeast selection cassette
- a CEN6/ARS4 yeast origin of replication
- an kanamycin E. coli selection casette
- a ColE1 E. coli origin of replication
- For each gene we targeted, we used Benchling to select 20-bp targeting sequences adjacent to PAMs. We attempted to find targeting sequences in the first 1/4 of the gene to ensure gene disruption.
- We designed and synthesized oligonucleotides as per the YTK instructions, annealed them, then used a GoldenGate reaction with Esp3I to clone them into the expression plasmid. We verified the plasmid using a traditional restriction map.
Construction of a URA3 knockout cassette
- We used Benchling to identify 40 bp upstream and downstream of the Cas9 target site.
- We designed primers against the URA3 selection cassette on plasmid YTK74 (from the Dueber lab's yeast toolkit) to append those 40 bp to cassette upstream and downstream.
- We synthesized those oligonucleotides, performed the PCR, verified the PCR product using gel electrophoresis and purified it using a silica spin column.
Transformation of S. cerevisiae
- We used Gietz' PEG/SS DNA/LiAc protocol to co-transform the Cas9/sgRNA plasmid and the URA3 cassette.
- We plated on leucine dropout plates to measure transformation efficiency, and uracil dropout plates to isolate clones in which the genes had been disrupted.