Abstract
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).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482(7385):331–338. https://doi.org/10.1038/nature10886
Hille F, Charpentier E (2016) CRISPR-Cas: biology, mechanisms and relevance. Philos Trans R Soc Lond Ser B Biol Sci 371(1707):20150496. https://doi.org/10.1098/rstb.2015.0496
Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A 109(39):E2579–E2586. https://doi.org/10.1073/pnas.1208507109
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. https://doi.org/10.1126/science.1225829
DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41(7):4336–4343. https://doi.org/10.1093/nar/gkt135
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.2015.02.001
Lobs AK, Engel R, Schwartz C, Flores A, Wheeldon I (2017) CRISPR-Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in Kluyveromyces marxianus. Biotechnol Biofuels 10:164. https://doi.org/10.1186/s13068-017-0854-5
Schwartz CM, Hussain MS, Blenner M, Wheeldon I (2016) Synthetic RNA polymerase III promoters facilitate high-efficiency CRISPR-Cas9-mediated genome editing in Yarrowia lipolytica. ACS Synth Biol 5(4):356–359. https://doi.org/10.1021/acssynbio.5b00162
Gao S, Tong Y, Wen Z, Zhu L, Ge M, Chen D, Jiang Y, Yang S (2016) Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system. J Ind Microbiol Biotechnol 43(8):1085–1093. https://doi.org/10.1007/s10295-016-1789-8
Jacobs JZ, Ciccaglione KM, Tournier V, Zaratiegui M (2014) Implementation of the CRISPR-Cas9 system in fission yeast. Nat Commun 5:5344. https://doi.org/10.1038/ncomms6344
Weninger A, Hatzl AM, Schmid C, Vogl T, Glieder A (2016) Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris. J Biotechnol 235:139–149. https://doi.org/10.1016/j.jbiotec.2016.03.027
Weninger A, Fischer J, Raschmanova H, Kniely C, Vogl T, Glieder A (2017) Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers. J Cell Biochem. https://doi.org/10.1002/jcb.26474
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/s12918-017-0492-3
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
Waldrip ZJ, Byrum SD, Storey AJ, Gao J, Byrd AK, Mackintosh SG, Wahls WP, Taverna SD, Raney KD, Tackett AJ (2014) A CRISPR-based approach for proteomic analysis of a single genomic locus. Epigenetics 9(9):1207–1211. https://doi.org/10.4161/epi.29919
Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5):1173–1183. https://doi.org/10.1016/j.cell.2013.02.022
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.2013.06.044
Farzadfard F, Perli SD, Lu TK (2013) Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS Synth Biol 2(10):604–613. https://doi.org/10.1021/sb400081r
Deaner M, Alper HS (2017) Systematic testing of enzyme perturbation sensitivities via graded dCas9 modulation in Saccharomyces cerevisiae. Metab Eng 40:14–22. https://doi.org/10.1016/j.ymben.2017.01.012
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/s13059-016-0900-9
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
Generoso WC, Gottardi M, Oreb M, Boles E (2016) Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae. J Microbiol Methods 127:203–205. https://doi.org/10.1016/j.mimet.2016.06.020
Gao Y, Zhao Y (2014) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J Integr Plant Biol 56(4):343–349. https://doi.org/10.1111/jipb.12152
Zhang XH, Tee LY, Wang XG, Huang QS, Yang SH (2015) Off-target effects in CRISPR/Cas9-mediated genome engineering. Mol Ther Nucleic Acids 4(11):e264. https://doi.org/10.1038/mtna.2015.37
O’Geen H, Yu AS, Segal DJ (2015) How specific is CRISPR/Cas9 really? Curr Opin Chem Biol 29:72–78. https://doi.org/10.1016/j.cbpa.2015.10.001
Jakociunas T, Bonde I, Herrgard M, Harrison SJ, Kristensen M, Pedersen LE, Jensen MK, Keasling JD (2015) Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae. Metab Eng 28:213–222. https://doi.org/10.1016/j.ymben.2015.01.008
Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3(11):e3647. https://doi.org/10.1371/journal.pone.0003647
Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4(5):e5553. https://doi.org/10.1371/journal.pone.0005553
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.3.1.18223
Sarkari P, Marx H, Blumhoff ML, Mattanovich D, Sauer M, Steiger MG (2017) An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger. Bioresour Technol 245(Pt B):1327–1333. https://doi.org/10.1016/j.biortech.2017.05.004
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-O
Gasser B, Prielhofer R, Marx H, Maurer M, Nocon J, Steiger M, Puxbaum V, Sauer M, Mattanovich D (2013) Pichia pastoris: protein production host and model organism for biomedical research. Future Microbiol 8:191–208. https://doi.org/10.2217/fmb.12.133
Wu S, Letchworth GJ (2004) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. BioTechniques 36(1):152–154
Hamilton S, Gerngross T (2007) Glycosylation engineering in yeast: the advent of fully humanized yeast. Curr Opin Biotechnol 18(5):387–392. https://doi.org/10.1016/j.copbio.2007.09.001
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
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.201600584
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Gassler, T., Heistinger, L., Mattanovich, D., Gasser, B., Prielhofer, R. (2019). CRISPR/Cas9-Mediated Homology-Directed Genome Editing in Pichia pastoris. In: Gasser, B., Mattanovich, D. (eds) Recombinant Protein Production in Yeast. Methods in Molecular Biology, vol 1923. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9024-5_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9024-5_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9023-8
Online ISBN: 978-1-4939-9024-5
eBook Packages: Springer Protocols