Difference between revisions of "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== | ==Resources== | ||
* Background strain: [https://www.atcc.org/en/Products/All/200897.aspx BY4735]. BY4735 is a [https://www.ncbi.nlm.nih.gov/pubmed/9483801 MATalpha 6-way auxotroph based on S288C], bearing non-revertable deletions in [[ADE2]], [[HIS3]], [[LEU2]], [[MET15]], [[TRP1]] and [[URA3]]. | * Background strain: [https://www.atcc.org/en/Products/All/200897.aspx BY4735]. BY4735 is a [https://www.ncbi.nlm.nih.gov/pubmed/9483801 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 [https://pubs.acs.org/doi/10.1021/sb500366v Dueber lab's yeast toolkit.] Additional assembly details are below. | * Plasmids: Knock-out plasmids were built using the [https://pubs.acs.org/doi/10.1021/sb500366v 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 [https://benchling.com/ 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. | ||
| + | |||
| + | ==Construction of a | ||
Revision as of 06:43, 30 April 2019
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
- 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.
==Construction of a
