Table 1.
Yeast strains used in this study.
| Strain | Genotype | References |
|---|---|---|
| BY4741 | MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | Brachmann et al., 1998 |
| BY4742 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 | Brachmann et al., 1998 |
| GFY-42 | BY4741; CDC10::mCherry::ADH(t)::SpHIS5 | Finnigan et al., 2015b |
| GFY-330 | BY4741; CDC12::GFP::ADH(t)::HygR + pCDC12::URA3 (pJT1622) | This study |
| GFY-1583 | BY4741; KEL1::TAPa::ADH(t)::SpHIS5 | TAP Tag Collection |
| GFY-1589 | BY4741; BUD3::TAPa::ADH(t)::SpHIS5 | TAP Tag Collection |
| GFY-1620 | BY4741; ELM1::TAPa::ADH(t)::SpHIS5 | TAP Tag Collection |
| GFY-2047 | BY4741; CAF120::TAPa::ADH(t)::SpHIS5 | TAP Tag Collection |
| GFY-2056 | BY4741; NBA1::TAPa::ADH(t)::SpHIS5 | TAP Tag Collection |
| GFY-2069 | BY4741; BEM2::TAPa::ADH(t)::SpHIS5 | TAP Tag Collection |
| GFY-2071 | BY4741; MYO4::TAPa::ADH(t)::SpHIS5 | TAP Tag Collection |
| GFY-2092 | BY4741; BUD2::TAPa::ADH(t)::SpHIS5 | TAP Tag Collection |
| GFY-2251b | BY4742; mso1Δ::KanR | Genome Deletion Collection |
| GFY-2259b | BY4742; bni4Δ::KanR | Genome Deletion Collection |
| GFY-2256b | BY4742; apl1Δ::KanR | Genome Deletion Collection |
| GFY-2613c | BY4741; his3Δ::prSHS1::GFP(S65T)::ADH(t)::KanR | This study |
| GFY-2615c, d | BY4741; his3Δ::prSHS1::eGFP::ADH(t)::KanR | This study |
| GFY-2617e | BY4741; his3Δ::prCDC12::GFP(S65T)::ADH(t)::KanR | This study |
| GFY-2621c | BY4741; his3Δ::prSHS1::mCherry::ADH(t)::KanR | This study |
| GFY-2622e | BY4741; his3Δ::prCDC12::mCherry::ADH(t)::KanR | This study |
| GFY-2440f | BY4741; cdc11Δ::cdc11(357–415Δ)::mCherry::CDC10 3′UTR::prGAL1/10::SpCas9::NLS::SHS1 3′UTR::prCCW12::KanR + (pGF-IVL1146; pRS316; prCDC11::CDC11(WT)) | This study |
| GFY-2442f | BY4742; cdc11Δ::cdc11(357–415Δ)::mCherry::CDC10 3′UTR::prGAL1/10::SpCas9::NLS::SHS1 3′UTR::prCCW12::KanR + (pGF-IVL1146; pRS316; prCDC11::CDC11(WT)) | This study |
| GFY-2625g | BY4741; cdc11Δ::CDC11(WT)::GFP::ADH(t)::SpHIS5 + (pSB1/JT1520; pRS316; prCDC11::CDC11(WT)) | This study |
| GFY-2624g | BY4742; cdc11Δ::CDC11(WT)::GFP::ADH(t)::SpHIS5 + (pSB1/JT1520; pRS316; prCDC11::CDC11(WT)) | This study |
The TAP (tandem affinity purification) tag consists of a linker sequence (11 residues), CBP domain (26), linker (9), TEV cleavage site (7), linker (10), first Protein Z domain (58), second Protein Z (58), and final linker (6). The two Protein Z domains are identical in sequence. All TAP-tag strains were tested as single clonal isolates; the genomic loci that were tagged were PCR amplified and confirmed via Sanger sequencing including (roughly) the last 200 bp of the tagged gene. The nine genes chosen occur on nine separate yeast chromosomes.
Strains from the haploid genome deletion collection were confirmed as single clonal isolates for resistance to G418 disulfide and proper knock-out of the intended gene by diagnostic PCRs.
Contains 449 bp of SHS1 5′ UTR. Strain GFY-2613 was constructed by PCR amplifying the prSHS1::GFP(S65T)::ADH(t)::KanR fragment from pGF-IVL1348 along with 500 bp of flanking homology engineered upstream of prSHS1 and downstream of the KanR cassette and transforming into WT BY4741 yeast. Strains GFY-2615, GFY-2621, and GFY-2622 were constructed in a similar manner from pGF-IVL1350, pGF-IVL1352, and pGF-IVL1353, respectively.
Enhanced GFP (eGFP) contains S65T, F64L, R88Q, and H239L.
Contains 477 bp of CDC12 5′ UTR.
The following strains were constructed by first adding pGF-IVL1146 to WT BY4741 (GFY-2442) or WT BY4742 (GFY-2440) yeast. This “covering vector” expresses WT CDC11 with 21 codons mutated from their native code to an alternative codon (without changing the final protein sequence). Second, the endogenous CDC11 was deleted by transforming this strain with a PCR fragment of cdc11Δ::HygR with flanking 5′ and 3′ UTR (300 bp) amplified from a chromosomal preparation of GFY-155 and selecting for resistance to hygromycin and lethality on media containing 5-FOA (loss of CDC11 renders cells inviable at 30°C). Third, a plasmid was constructed using three rounds of subsequent in vivo ligation and homologous recombination in yeast (Finnigan and Thorner, 2015) to assemble prCDC11::cdc11(357-415Δ)::mCherry::CDC10 3′UTR::prGAL1/10::SpCas9::NLS::SHS1 3′UTR::prCCW12::KanR on pRS315 (pGF-IVL1149). S. pyogenes Cas9 (yeast codon bias, CAI = 0.92) contains a C-terminal SV40 nuclear localization signal (SRADPKKKRKV) and is under transcriptional control of the GAL1/10 promoter (814 bp). The cdc11(357-415Δ) mutant contains 13 residues with alternative codons near the N-terminus and contains the CDC10 terminator sequence (465 bp). Cas9 contains the SHS1 terminator sequence (486 bp). The KanR MX cassette has been modified to remove the P(tef) constitutive promoter with the native yeast CCW12 promoter (992 bp) which still allows for selection of resistance to G418 disulfide. This entire linear fragment containing both CDC11 and Cas9 was amplified in 2 separate PCR reactions with overlapping sequence within the central region of Cas9 and co-transformed into the cdc11Δ::HygR yeast also expressing the aforementioned covering vector. The CDC11 promoter (330 bp) and MX(terminator) sequence (235 bp) provided homology to the native CDC11 locus to integrate the entire gene drive cassette. The Cdc11 C-Terminal Extension domain (residues 357-415; CTE) is not required for septin filament formation (Versele et al., 2004) yet causes a severe loss of function when paired with shs1Δ or other mutations such as bni5Δ (Finnigan et al., 2015a).
GFY-150 yeast (BY4742; cdc11Δ::KanR) or GFY-153 (BY4741; cdc11Δ::KanR) were transformed with a PCR fragment containing prCDC11::CDC11(WT)::GFP::ADH(t)::SpHIS5 (amplified from pGF-IVL1354) to create GFY-2624 and GFY-2625.