Preloading budding yeast with all-in-one CRISPR/Cas9 vectors for easy and high-efficient genome editing
Main Article Content
Keywords
all-in-one, CRISPR/Cas9, inducible, mating type switching, multiplex genome editing, Saccharomyces cerevisiae
Abstract
The CRISPR/Cas9 technology has greatly improved genome editing in Saccharomyces cerevisiae over recent years. However, several current CRISPR/Cas9 systems suffer from work-intensive cloning procedures and/or the requirement of co-transforming target cells with multiple system components simultaneously which can reduce the effectivity of such applications. Here, we present a new set of all-in-one CRISPR/Cas9 vectors that combine unique benefits of different already existent systems in order to further expand the technology’s design possibilities. Our vectors mediate constitutive gRNA expression whereas Cas9 expression is either driven from a constitutive or an inducible promoter. The introduction of desired gRNA targeting sequences into our inducible single gRNA vector relies just on in vivo homologous recombination-mediated assembly of overlapping single-stranded oligonucleotides, thus reducing efforts of plasmid cloning to an absolute minimum. By employing the inducible system, yeast cells can be easily preloaded with plasmids encoding for a functional CRISPR/Cas9 system, thereby chronologically separating the cloning procedure from the genome editing step. Gene knockouts could be achieved with high efficiency and effectivity by simply transforming preloaded cells with a selectable disruption cassette without the need of co-introducing any CRISPR/Cas9 system component. We also show the feasibility of efficient gene knockouts even when multiple gene copies were present such as in non-haploid strain backgrounds as well as the simultaneous deletion of two different genes in a haploid genetic background by using a multiplex variant of our inducible vector. The versatile applicability of our inducible vector system was further demonstrated by CRISPR/Cas9-mediated mating type switching of yeast.
Downloads
Download data is not yet available.
Metrics
Metrics Loading ...
References
1. Stovicek V, Holkenbrink C, Borodina I (2017) CRISPR/Cas system for yeast genome engineering: advances and applications. FEMS Yeast Res.17(5):1-7.
2. Mans R, van Rossum HM, Wijsman M, Backx A, Kuijpers NG, et al. (2015) CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Res 15(2):1-15.
3. Ryan OW, Cate JH (2014) Multiplex engineering of industrial yeast genomes using CRISPRm. Methods Enzymol 546:473-89.
4. Generoso WC, Gottardi M, Oreb M, Boles E (2016) Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae. J Microbiol Methods 127:203-5.
5. Hu G, Luo S, Rao H, Cheng H, Gan X (2018) A Simple PCR-based Strategy for the Introduction of Point Mutations in the Yeast Saccharomyces cerevisiae via CRISPR/Cas9. Biochem Mol Biol J 4(1):9.
6. Reider Apel A, d'Espaux L, Wehrs M, Sachs D, Li RA, et al. (2017) A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. Nucleic Acids Res 45(1):496-508.
7. Bao Z, Xiao H, Liang J, Zhang L, Xiong X, et al. (2015) Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae. ACS Synth Biol 4(5):585-94.
8. Laughery MF, Hunter T, Brown A, Hoopes J, Ostbye T, et al. (2015) New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. Yeast 32(12):711-20.
9. Gibson DG (2009) Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides. Nucleic Acids Res 37(20):6984-90.
10. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, et al. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14(2):115-32.
11. Entian K-D, Kötter P (2007) 25 Yeast Genetic Strain and Plasmid Collections. Methods in Microbiology 36:629-66.
12. Mirzayan C, Copeland CS, Snyder M (1992) The NUF1 gene encodes an essential coiled-coil related protein that is a potential component of the yeast nucleoskeleton. J Cell Biol 116(6):1319-32.
13. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, et al. (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285(5429):901-6.
14. Jakociunas T, Rajkumar AS, Zhang J, Arsovska D, Rodriguez A, et al. (2015) CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae. ACS Synth Biol 4(11):1226-34.
15. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, et al. (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41(7):4336-43.
16. Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH (2002) A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res 30(6):e23.
17. Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24(13):2519-24.
18. Jansen G, Wu C, Schade B, Thomas DY, Whiteway M (2005) Drag&Drop cloning in yeast. Gene 344:43-51.
19. Huxley C, Green ED, Dunham I (1990) Rapid assessment of S. cerevisiae mating type by PCR. Trends Genet 6(8):236.
20. Da Silva NA, Srikrishnan S (2012) Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae. FEMS Yeast Res 12(2):197-214.
21. Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197(2):345-6.
22. Ugolini S, Bruschi CV (1996) The red/white colony color assay in the yeast Saccharomyces cerevisiae: epistatic growth advantage of white ade8-18, ade2 cells over red ade2 cells. Curr Genet 30(6):485-92.
23. Frankenberg-Schwager M, Frankenberg D (1990) DNA double-strand breaks: their repair and relationship to cell killing in yeast. Int J Radiat Biol 58(4):569-75.
24. Weiffenbach B, Haber JE (1981) Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae. Mol Cell Biol 1(6):522-34.
25. Mortimer RK (2000) Evolution and variation of the yeast (Saccharomyces) genome. Genome Res 10(4):403-9.
26. Replansky T, Koufopanou V, Greig D, Bell G (2008) Saccharomyces sensu stricto as a model system for evolution and ecology. Trends Ecol Evol 23(9):494-501.
27. Mortimer RK, Romano P, Suzzi G, Polsinelli M (1994) Genome renewal: a new phenomenon revealed from a genetic study of 43 strains of Saccharomyces cerevisiae derived from natural fermentation of grape musts. Yeast 10(12):1543-52.
28. Storchova Z (2014) Ploidy changes and genome stability in yeast. Yeast 31(11):421-30.
29. Koopman F, Beekwilder J, Crimi B, van Houwelingen A, Hall RD, et al. (2012) De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae. Microb Cell Fact 11:155.
30. Zhang GC, Kong, II, Kim H, Liu JJ, Cate JH, et al. (2014) Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease. Appl Environ Microbiol 80(24):7694-701.
31. Meiron H, Nahon E, Raveh D (1995) Identification of the heterothallic mutation in HO-endonuclease of S. cerevisiae using HO/ho chimeric genes. Curr Genet 28(4):367-73.
32. Fukuda N, Matsukura S, Honda S (2013) Artificial conversion of the mating-type of Saccharomyces cerevisiae without autopolyploidization. ACS Synth Biol 2(12):697-704.
33. Herskowitz I, Jensen RE (1991) Putting the HO gene to work: practical uses for mating-type switching. Methods Enzymol 194:132-46.
34. Xie ZX, Mitchell LA, Liu HM, Li BZ, Liu D, et al. (2018) Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of Saccharomyces cerevisiae. G3 (Bethesda) 8(1):173-83.
2. Mans R, van Rossum HM, Wijsman M, Backx A, Kuijpers NG, et al. (2015) CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Res 15(2):1-15.
3. Ryan OW, Cate JH (2014) Multiplex engineering of industrial yeast genomes using CRISPRm. Methods Enzymol 546:473-89.
4. Generoso WC, Gottardi M, Oreb M, Boles E (2016) Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae. J Microbiol Methods 127:203-5.
5. Hu G, Luo S, Rao H, Cheng H, Gan X (2018) A Simple PCR-based Strategy for the Introduction of Point Mutations in the Yeast Saccharomyces cerevisiae via CRISPR/Cas9. Biochem Mol Biol J 4(1):9.
6. Reider Apel A, d'Espaux L, Wehrs M, Sachs D, Li RA, et al. (2017) A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. Nucleic Acids Res 45(1):496-508.
7. Bao Z, Xiao H, Liang J, Zhang L, Xiong X, et al. (2015) Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae. ACS Synth Biol 4(5):585-94.
8. Laughery MF, Hunter T, Brown A, Hoopes J, Ostbye T, et al. (2015) New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. Yeast 32(12):711-20.
9. Gibson DG (2009) Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides. Nucleic Acids Res 37(20):6984-90.
10. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, et al. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14(2):115-32.
11. Entian K-D, Kötter P (2007) 25 Yeast Genetic Strain and Plasmid Collections. Methods in Microbiology 36:629-66.
12. Mirzayan C, Copeland CS, Snyder M (1992) The NUF1 gene encodes an essential coiled-coil related protein that is a potential component of the yeast nucleoskeleton. J Cell Biol 116(6):1319-32.
13. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, et al. (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285(5429):901-6.
14. Jakociunas T, Rajkumar AS, Zhang J, Arsovska D, Rodriguez A, et al. (2015) CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae. ACS Synth Biol 4(11):1226-34.
15. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, et al. (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41(7):4336-43.
16. Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH (2002) A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res 30(6):e23.
17. Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24(13):2519-24.
18. Jansen G, Wu C, Schade B, Thomas DY, Whiteway M (2005) Drag&Drop cloning in yeast. Gene 344:43-51.
19. Huxley C, Green ED, Dunham I (1990) Rapid assessment of S. cerevisiae mating type by PCR. Trends Genet 6(8):236.
20. Da Silva NA, Srikrishnan S (2012) Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae. FEMS Yeast Res 12(2):197-214.
21. Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197(2):345-6.
22. Ugolini S, Bruschi CV (1996) The red/white colony color assay in the yeast Saccharomyces cerevisiae: epistatic growth advantage of white ade8-18, ade2 cells over red ade2 cells. Curr Genet 30(6):485-92.
23. Frankenberg-Schwager M, Frankenberg D (1990) DNA double-strand breaks: their repair and relationship to cell killing in yeast. Int J Radiat Biol 58(4):569-75.
24. Weiffenbach B, Haber JE (1981) Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae. Mol Cell Biol 1(6):522-34.
25. Mortimer RK (2000) Evolution and variation of the yeast (Saccharomyces) genome. Genome Res 10(4):403-9.
26. Replansky T, Koufopanou V, Greig D, Bell G (2008) Saccharomyces sensu stricto as a model system for evolution and ecology. Trends Ecol Evol 23(9):494-501.
27. Mortimer RK, Romano P, Suzzi G, Polsinelli M (1994) Genome renewal: a new phenomenon revealed from a genetic study of 43 strains of Saccharomyces cerevisiae derived from natural fermentation of grape musts. Yeast 10(12):1543-52.
28. Storchova Z (2014) Ploidy changes and genome stability in yeast. Yeast 31(11):421-30.
29. Koopman F, Beekwilder J, Crimi B, van Houwelingen A, Hall RD, et al. (2012) De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae. Microb Cell Fact 11:155.
30. Zhang GC, Kong, II, Kim H, Liu JJ, Cate JH, et al. (2014) Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease. Appl Environ Microbiol 80(24):7694-701.
31. Meiron H, Nahon E, Raveh D (1995) Identification of the heterothallic mutation in HO-endonuclease of S. cerevisiae using HO/ho chimeric genes. Curr Genet 28(4):367-73.
32. Fukuda N, Matsukura S, Honda S (2013) Artificial conversion of the mating-type of Saccharomyces cerevisiae without autopolyploidization. ACS Synth Biol 2(12):697-704.
33. Herskowitz I, Jensen RE (1991) Putting the HO gene to work: practical uses for mating-type switching. Methods Enzymol 194:132-46.
34. Xie ZX, Mitchell LA, Liu HM, Li BZ, Liu D, et al. (2018) Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of Saccharomyces cerevisiae. G3 (Bethesda) 8(1):173-83.
Article Sidebar
Published
Sep 12, 2018
Article Details
How to Cite
1.
Degreif D, Kremenovic M, Geiger T, Bertl A. Preloading budding yeast with all-in-one CRISPR/Cas9 vectors for easy and high-efficient genome editing. J Biol Methods [Internet]. 2018 Sep. 12 [cited 2024 Apr. 26];5(3):e98. Available from: https://jbmethods.org/index.php/jbm/article/view/254
Issue
Section
Articles
Authors who publish with JBM agree to the following terms:
- Authors retain copyright and grant JBM right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
Author Biography
Daniel Degreif, Technische Universität Darmstadt, FB Biology; Yeast Membrane Biology
Department of Biology, Yeast Membrane Biology PostDoc
