Table 2.
List of available CRISPR/Cas9 tools for yeast.
| Reference | Availability | Organism and strain (ploidy) | Cas9 expression (vector, selection marker, promoter) | gRNA expression (vector, selection marker, promoter, terminator) | Application and efficiency |
|---|---|---|---|---|---|
| S. cerevisiae genome editing | |||||
| DiCarlo et al. (2013) | Addgene | S. cerevisiae BY4733 (n) | CEN/ARS, TRP1, PTEF1/GALL-Cas9 | 2μ, URA3, PSNR52/TSUP4 | Single-gene disruption/marker cassette insertion: 99% |
| Gao and Zhao (2014) | Addgene | S. cerevisiae LPY16936 (n) | 2μ, LEU2, PADH1-Cas9 | 2μ, URA3, PADH1-HH ribozyme/HDV ribozyme-TADH1 | Single-gene disruption: 100% |
| Ryan et al. (2014) | On request | S. cerevisiae S288C (n, 2n) ATCC4124 (poly n) | a 2μ, kanMX, PRNR2-Cas9b | tRNAPro -HDV ribozyme/TSNR52 | Single/multiple_gene disruption(s): 90%–100%/19%–85%, three-part marker cassette insertion: 70%–85% |
| Bao et al. (2015) | Addgene | S. cerevisiae BY4741 (n) CEN.PK2–1c (n) | a 2μ, truncated URA3, PTEF1-iCas9b | PSNR52-crRNA/TSUP4, PRPR1-tracrRNA/TRPR1 | Single/multiple-gene disruption: 27%–100% |
| Zhang et al. (2014) | Addgene | S. cerevisiae ATCC 4124 (poly n) | CEN/ARS, natMX, PTEF1-Cas9 | 2μ, hphMX, PSNR52/TSUP4 | Single-gene disruption: 15%–60% |
| Jakočiūnas et al. (2015a) | On request | S. cerevisiae CEN.PK2–1c (n) | CEN/ARS, TRP1, PTEF1-Cas9 | 2μ, LEU2, PSNR52/TSUP4 | Single/multiple-gene disruption(s): 100%/50%–100% |
| Mans et al. (2015) | Euroscarf | S. cerevisiae CEN.PK2–1c (n) CEN.PK113–7D (n) CEN.PK122 (2n) | integr. can1Δ::PTEF1-Cas9-natMX | 2μ, URA3, amdSYM/hphMX/ kanMX/LEU2/natMX/ HIS3/ TRP1, PSNR52/TSUP4 | Single-gene deletion: 25%–75%, multiple-gene deletions/multiple-gene cassette insertions: 65%–100% |
| Stovicek, Borodina and Forster (2015) | Addgene | S. cerevisiae CEN.PK113–7D (n) Ethanol Red, | CEN/ARS, kanMX, PTEF1/ADH1-Cas9 | 2μ, natMX, PSNR52/TSUP4 | Single-gene disruption and gene cassette insertion: 65%–97% |
| CLIB382, CBS7960 (2n) | |||||
| Horwitz et al. (2015) | On request | S. cerevisiae CEN.PK2–1c (n) | integr. gre3Δ::PFBA1-Cas9c-hphMX | 2μ, URA3/HIS3/natMX, PSNR52/TSUP4 | Single allele swap: 82%–100%, multiple-gene disruptions: 65%–91%, multiple-gene cassette integrations: 4.2% |
| Tsai et al. (2015) | On request | S. cerevisiae D452–2 (n) | CEN/ARS, natMX, PTEF1-Cas9 | 2μ, hphMX, PSNR52/TSUP4 | Two part-gene cassettes integration into a single-gene locus: 25%–100% |
| Laughery et al. (2015) | Addgene | S. cerevisiae BY4741 (n) | a 2μ, LEU2/URA3, PTDH3-Cas9 | PSNR52/TSUP4 | Single-gene disruption: 97%–98% |
| Lee et al. (2015) | Addgene | S. cerevisiae S288C (n) | a CEN/ARS, URA3, PPGK1-Cas9 | tRNAPhe-HDV ribozyme/TSNR52 | Single/multiple-gene disruption(s): 96%/21%–76% |
| Jakočiūnas et al. (2015b) | On request | S. cerevisiae CEN.PK111−27B (n) | CEN/ARS, TRP1, PTEF1-Cas9 | 2μ, LEU2, PSNR52/TSUP4 | Multiple part gene cassette integrations into multiple gene loci: 30%–97% |
| Ronda et al. (2015) | On request | S. cerevisiae CEN.PK2–1c (n) | CEN/ARS, TRP1, PTEF2-Cas9 | 2μ, natMX, PSNR52/TSUP4 | Gene cassette integration into multiple intergenic loci: 84–100% |
| Shi et al. (2016) | On request | S. cerevisiae HZ848 (n), CEN.PK2–1c (n) | a 2μ, truncated URA3, PTEF1-Cas9 | PSNR52-crRNA/TSUP4, PRPR1-tracrRNA/TRPR1 | Long gene fragment integration into multiple genomic loci: 75%–88% |
| Generoso et al. (2016) | Addgene | S. cerevisiae CEN.PK113–7D (n) Ethanol Red (2n) | a 2μ, kanMX/natMX, PROX3-Cas9c | PSNR52/TSUP4 | Single and double-gene disruptions: 91%–98% |
| Jessop-Fabre et al. (2016) | Addgene | S. cerevisiae CEN.PK113–7D (n) Ethanol Red (2n) | CEN/ARS, kanMX, PTEF1-Cas9 | 2μ, natMX, PSNR52/TSUP4 | Integration of a long gene fragment into a single locus: 95%–100%/multiple loci: 60–70% |
| Reider Apel et al. (2016) | On request | S. cerevisiae BY4742 (n) | a 2μ, URA3, LEU2, PADH1-Cas9c | tRNATyr -HDV ribozyme/TSNR52 | Three part-gene cassette integration into mutiple intergenic loci: 40%–95% |
| Garst et al. (2017) | On request | S. cerevisiae | a 2μ, truncated URA3, PTEF1-iCas9b | PSNR52-crRNA/TSUP4, PRPR1-tracrRNA/TRPR1 | Single-gene non-sense mutation: 70%–95% |
| BY4709 (n) | |||||
| RM11–1 (n) | |||||
| Liu et al. (2017) | On request | S. cerevisiae var. boulardii ATCC MYA-796 (n) | CEN/ARS, natMX, PTEF1-Cas9 | 2μ, hphMX, PSNR52/TSUP4 | Single/double-gene disruption(s):100%/N/A |
| Nishida et al. (2016) | Addgene | S. cerevisiae | CEN/ARS, LEU2, PGAL1-nCas9 (840A)/nCas9 (D10A)/n(d)Cas9 (D10A/840A)-PmCDA1 | 2μ, URA3, PSNR52/TSUP4 | Cytidine deaminase-mediated single/double-gene disruption(s): 16%–54%/14%–31% |
| BY4741 (n) | |||||
| YPH501 (2n) | |||||
| Vanegas, Lehka and Mortensen (2017) | On request | S. cerevisiaeS288C (n)PJ69–4 (n) | Integr. intergenic X-3::PTEF1-Cas9-URA3 | CEN/ARS, LEU2PSNR52/TSUP4 | Integration of three-part multiple-gene fragment into an intergenic site: 100% |
| S. cerevisiae gene activation/repression | |||||
| Gilbert et al. (2013) | Addgene | S. cerevisiaeW303 | CEN/ARS, LEU2, PTDH3-dCas9(-Mxi1) | CEN/ARS, URA3, PSNR52/TSUP4 | Several 10-fold reporter gene transcription repression (CRISPRi) |
| Farzadfard, Perli and Lu (2013) | Addgene | S. cerevisiaeW303 (n) | integr. Δtrp:: PTPGI-dCas9c-VP64-TRP1 | 2μ, HIS3/LEU2, PRPR1/TRPR1 | Transcription activation (activator domain)/repression (CRISPRi) |
| Zalatan et al. (2015) | Addgene | S. cerevisiaeW303 (n) | integr. Δleu2/his3::PTDH3/GAL10-dCas9-CgLEU2/HIS3 | CEN/ARS, URA3, PSNR52-gRNA-sc(scaffold)RNA/TSUP4 | Multiple-gene transcription activation (RNA-binding chimeric activators)/repression (CRISPRi) |
| Chavez et al. (2015) | Addgene | S. cerevisiaeW303 (n) | CEN/ARS, TRP1, PTDH3-dCas9-VPR | 2μ, URA3, PSNR52/TSUP4 | Transcription activation (multiple activation domains) |
| Smith et al. (2016) | Addgene | S. cerevisiaeBY4741 (n) | a CEN/ARS, TRP1/URA3, PTEF1-dCas9-Mxi1 | PRPR1(TetO)/TRPR1 | Transcription repression (repression domain- CRISPRi) |
| Vanegas, Lehka and Mortensen (2017) | On request | S. cerevisiaeS288C (n)PJ69–4 (n) | Integr. Intergenic X-3::PTEF1-dCas9c/dCas9-VP64-URA3 | CEN/ARS, LEU2PSNR52/TSUP4 | Transcription activation (activator domain)/repression (CRISPRi) |
| Deaner and Alper (2017) | On request | S. cerevisiaeBY4741 (n) | a CEN/ARS, LEU2, PTDH3-dCas9-Mxi1/PTDH3-dCas9-VPR | PSNR52/TSUP4 | Graded gene activation/repression (fold transcription activation-gene silencing) |
| K. lactis | |||||
| Horwitz et al. (2015) | On request | K. lactis ATCC8585 (n) | integr. Klgal80::PFBA1-Cas9c-hphMX | pKD1, natMX, PSNR52/TSUP4 | Multiple-gene cassette insertion into multiple-gene loci: 2.1% |
| Y. lipolytica | |||||
| Schwartz et al. (2016a) | Addgene | Y. lipolytica ATCC MYA-2613 (n) | a CEN, LEU2, PUAS1B8-TEF-Cas9c | SCR'-tRNAGly/polyT | Single-gene disruptions (NHEJ/HR): 90%–100%/64%–88% (100% in KU mutant) |
| Gao et al. (2016) | Addgene | Y. lipolytica ATCC 201 249 ATCC MYA-2613 (n) | a CEN, LEU2/URA3, PTEFin-Cas9 | PTEFin-HH ribozyme/HDV ribozyme-TMIG1 | Single-gene disruption (NHEJ/HR): 62%–98%/72% (94% in KU mutant), multiple-gene disruptions (NHEJ): 19%–37% |
| Schwartz et al. (2016b) | Addgene | Y. lipolytica ATCC MYA-2613 (n) | a CEN, LEU2, PUAS1B8-TEF-Cas9c | SCR'-tRNAGly/polyT | Gene cassette integration into an intergenic locus: 48%–69% |
| Ko. phaffii (P. pastoris) | |||||
| Weninger et al. (2016) | On request | Ko. phaffii (P. pastoris) CBS7435 (n) | PARS1a, ZEO, PHTX1-Cas9 | PHTX1-HH ribozyme/HDV ribozyme-TAOX1 | Single-gene disruption: 87%–94%, double-gene disruptions: 69% |
| Sch. pombe | |||||
| Jacobs et al. (2014) | Addgene | Sch. pombe (n) | a ars, ura4, Padh1-Cas9 | Prrk1/HH ribozyme-Trrk1 | Single-gene disruption (allele swap): 85%–90% |
| Fernandez and Berro (2016) | On request | Sch. pombe FY527 (n) FY528 (n) | a ars, ura4/fex1, Padh1-Cas9 | Prrk1/HH ribozyme-Trrk1 | Single-gene deletion (ORF removal): 33% |
| C. albicans | |||||
| Vyas, Barrasa and Fink (2015) | On request | C. albicans SC5314 (2n) | a integr. ENO1 locus, natMX, PENO1-Cas9c | PSNR52/TENO1 | Single/multiple gene disruption(s): 60%–80%/20% |
| Min et al. (2016) | On request | C. albicans SC5314 (2n) SN152 (2n) | linear cassette, PENO1-Cas9c | linear cassette, PSNR52/TENO1 | Single-gene deletion (ORF replacement with marker cassette): 45%–67% |
| C. glabrata | |||||
| Enkler et al. (2016) | On request | C. glabrata CBS138 (n) | CEN, TRP1, PCgCYC1-Cas9 | CEN, LEU2, PCgRNAH1/TCgTY2 | Single-gene disruption |
| Cr. neoformans | |||||
| Wang et al. (2016) | On request | Cr. neoformans serotype D strain JEC21 (n) | a linear vector, URA5, PACT1-Cas9 | URA5, PCnU6/polyT | Single-gene disruption (NHEJ/HR): 40%–90%/20%–90% |
| Arras et al. (2016) | On request | Cr. neoformans serotype A strain H99 (n) | integr. ‘Safe Heaven’-PTEF1-Cas9 | linear vector, PACT1-HH ribozyme/HDV ribozyme-TTRP1 | Single-gene deletion (ORF replacement with marker cassette): 65%–70% |
The Cas9 gene is a human codon-optimized version unless otherwise marked. Addgene CRISPR/Cas9 plasmids for use in yeast are available at https://www.addgene.org/crispr/yeast/. Euroscarf deposited vectors can be ordered here www.euroscarf.de.
HH—Hammerhead ribozyme, HDV—hepatitis delta virus ribozyme, iCas9 – mutated ‘hyperactive’ variant, nCas9 – mutated ‘nicking’ variant causing single-strand DNA break, dCas9 – ‘dead’ nuclease activity-lacking variant, PmCDA1 – cytidine deaminase from sea lamprey (Petromyzon marinus), Mxi1 – mammalian transcriptional repressor, VP64 – mammalian transcriptional activator domain, VPR—VP64-p65-Rta tripartite activator domain.
a
Both components on a single expression element.
b
Native S. pyogenes Cas9.
c
Species codon-optimized Cas9.