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 the URA3 selection cassette on plasmid YTK74 (from the Dueber lab's yeast toolkit) to append those 40 bp to cassette upstream and downstream.
3. 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

1. We used Gietz' PEG/SS DNA/LiAc protocol to co-transform the Cas9/sgRNA plasmid and the URA3 cassette.
2. We plated on leucine dropout plates to measure transformation efficiency, and uracil dropout plates to isolate clones in which the genes had been disrupted.

Verification of gene disruption

1. We used a lithium acetate / SDS method to extract genomic DNA from putative knockout strains
2. We used Benchling to design PCR primers upstream and downstream of the knockout site.
3. We used PCR followed by gel electrophoresis to determine if the URA3 cassette had inserted at the desired site.