UW-Stout/Knockout Protocol

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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.

Resources

• 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

1. 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
2. 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.
3. 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

1. We used Benchling to identify 40 bp upstream and downstream of the Cas9 target site.
2. We designed primers against YTK74