CRISPR/Cas9-Mediated Homology-Directed Genome Editing in Pichia pastoris
State-of-the-art strain engineering techniques for the methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) include overexpression of endogenous and heterologous genes and deletion of host genes. For efficient gene deletion, methods such as the split-marker technique have been established. However, synthetic biology trends move toward building up large and complex reaction networks, which often require endogenous gene knockouts and simultaneous overexpression of individual genes or whole pathways. Realization of such engineering tasks by conventional approaches employing subsequent steps of transformations and marker recycling is very time- and labor-consuming. Other applications require tagging of certain genes/proteins or promoter exchange approaches, which are hard to design and construct with conventional methods. Therefore, efficient systems are required that allow precise manipulations of the P. pastoris genome, including simultaneous overexpression of multiple genes. To meet this challenge, we have developed a CRISPR/Cas9-based kit for gene insertions, deletions, and replacements, which paves the way for precise genomic modifications in P. pastoris. In this chapter, the versatile method for performing these modifications without the integration of a selection marker is described. A ready-to-use plasmid kit for performing CRISPR/Cas9-mediated genome editing in P. pastoris based on the GoldenPiCS modular cloning vectors is available at Addgene as CRISPi kit (#1000000136).
Key wordsPichia pastoris Synthetic biology Genome editing GoldenPiCS CRISPR/Cas9
This work was supported by the Federal Ministry for Digital and Economic Affairs (BMDW), the Federal Ministry of Traffic, Innovation and Technology (BMVIT), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, the Government of Lower Austria and ZIT-Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG; and the Austrian Federal Ministry for Digital and Economic Affairs (BMDW), the National Foundation for Research, Technology and Development and the Christian Doppler Research Association. TG and LH were supported by the Austrian Science Fund (FWF): Doctoral Program BioToP—Biomolecular Technology of Proteins (FWF W1224). We further want to thank Franz Zehetbauer and Dariusz Yarych for technical support as well as Corinna Rebnegger and Matthias Steiger for initial inspiration and fruitful discussions.
- 6.Horwitz AA, Walter JM, Schubert MG, Kung SH, Hawkins K, Platt DM, Hernday AD, Mahatdejkul-Meadows T, Szeto W, Chandran SS, Newman JD (2015) Efficient multiplexed integration of synergistic alleles and metabolic pathways in yeasts via CRISPR-Cas. Cell Syst 1(1):88–96. https://doi.org/10.1016/j.cels.2015.02.001CrossRefPubMedGoogle Scholar
- 13.Prielhofer R, Barrero JJ, Steuer S, Gassler T, Zahrl R, Baumann K, Sauer M, Mattanovich D, Gasser B, Marx H (2017) GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris. BMC Syst Biol 11(1):123. https://doi.org/10.1186/s12918-017-0492-3CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Heistinger L, Gasser B, Mattanovich D (2018) Creation of stable heterothallic strains of Komagataella phaffii enables dissection of mating gene regulation. Mol Cell Biol 38(2). https://doi.org/10.1128/mcb.00398-17
- 17.Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154(2):442–451. https://doi.org/10.1016/j.cell.2013.06.044CrossRefPubMedPubMedCentralGoogle Scholar
- 20.Smith JD, Suresh S, Schlecht U, Wu M, Wagih O, Peltz G, Davis RW, Steinmetz LM, Parts L, St Onge RP (2016) Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design. Genome Biol 17:45. https://doi.org/10.1186/s13059-016-0900-9CrossRefPubMedPubMedCentralGoogle Scholar
- 21.Ryan OW, Skerker JM, Maurer MJ, Li X, Tsai JC, Poddar S, Lee ME, DeLoache W, Dueber JE, Arkin AP, Cate JH (2014) Selection of chromosomal DNA libraries using a multiplex CRISPR system. elife 3. https://doi.org/10.7554/eLife.03703
- 29.Werner S, Engler C, Weber E, Gruetzner R, Marillonnet S (2012) Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Bioeng Bugs 3(1):38–43. https://doi.org/10.1371/journal.pone.001676510.4161/bbug.3.1.18223CrossRefPubMedGoogle Scholar
- 31.Fairhead C, Llorente B, Denis F, Soler M, Dujon B (1996) New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using ‘split-marker’ recombination. Yeast 12(14):1439–1457. https://doi.org/10.1002/(SICI)1097-0061(199611)12:14<1439::AID-YEA37>3.0.CO;2-OCrossRefPubMedGoogle Scholar
- 35.Stovicek V, Holkenbrink C, Borodina I (2017) CRISPR/Cas system for yeast genome engineering: advances and applications. FEMS Yeast Res 17(5). https://doi.org/10.1093/femsyr/fox030
- 36.Marsalek L, Gruber C, Altmann F, Aleschko M, Mattanovich D, Gasser B, Puxbaum V (2017) Disruption of genes involved in CORVET complex leads to enhanced secretion of heterologous carboxylesterase only in protease deficient Pichia pastoris. Biotechnol J 12(5). https://doi.org/10.1002/biot.201600584CrossRefGoogle Scholar