Abstract
Pichia pastoris has gained much attention as a popular microbial cell factory for the production of recombinant proteins and high-value chemicals from laboratory to industrial scale. However, the lack of convenient and efficient genome engineering tools has impeded further applications of Pichia pastoris towards metabolic engineering and synthetic biology. Here, we report a CRISPR-based toolbox for gene editing and transcriptional regulation in P. pastoris. Based on the previous attempts in P. pastoris, we constructed a CRISPR/Cas9 system for gene editing using the RNA Pol-III–driven expression of sgRNA. The system was used to rapidly recycle the selectable marker with an eliminable episomal plasmid and achieved up to 100% knockout efficiency. Via dCas9 fused with transcriptional repressor (Mix1/RD1152) or activator (VPR), a flexible toolbox for regulation of gene expression was developed. The reporter gene eGFP driven by yeast pGAP or pCYC1 promoter showed strong inhibition (above 70%) and up to ~ 3.5-fold activation. To implement the combinatorial genetic engineering strategy, the CRISPR system contained a single Cas9-VPR protein, and engineered gRNA was introduced in P. pastoris for simultaneous gene activation, repression, and editing (CRISPR-ARE). We demonstrated that CRISPR-ARE was highly efficient for eGFP activation, mCherry repression, and ADE2 disruption, individually or in a combinatorial manner with a stable expression of multiplex sgRNAs. The simple and multifunctional toolkit demonstrated in this study will accelerate the application of P. pastoris in metabolic engineering and synthetic biology.
Key points
• An eliminable CRISPR/Cas9 system yielded a highly efficient knockout of genes.
• Simplified CRISPR/dCas9-based tools enabled transcriptional regulation of targeted genes.
• CRISPR-ARE system achieved simultaneous gene activation, repression, and editing in P. pastoris.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
All data generated or analyzed during this study are included in this published article (and its supplementary information files).
References
Anzalone AV, Koblan LW, Liu DR (2020) Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol 38:824–844. https://doi.org/10.1038/s41587-020-0561-9
Baumschabl M, Prielhofer R, Mattanovich D, Steiger MG (2020) Fine-tuning of transcription in Pichia pastoris using dCas9 and RNA scaffolds. ACS Synth Biol 9:3202–3209. https://doi.org/10.1021/acssynbio.0c00214
Dahlman JE, Abudayyeh OO, Joung J, Gootenberg JS, Zhang F, Konermann S (2015) Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat Biotechnol 33:1159–1161. https://doi.org/10.1038/nbt.3390
Dalvie NC, Leal J, Whittaker CA, Yang Y, Brady JR, Love KR, Love JC (2020) Host-Informed Expression of CRISPR Guide RNA for Genomic Engineering in Komagataella Phaffii. ACS Synth Biol 9:26–35. https://doi.org/10.1021/acssynbio.9b00372
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 41:4336–4343. https://doi.org/10.1093/nar/gkt135
Dong C, Jiang L, Xu S, Huang L, Cai J, Lian J, Xu Z (2020) A single Cas9-VPR nuclease for simultaneous gene activation, repression, and editing in Saccharomyces cerevisiae. ACS Synth Biol 9:2252–2257. https://doi.org/10.1021/acssynbio.0c00218
Fischer JE, Glieder A (2019) Current advances in engineering tools for Pichia pastoris. Curr Opin Biotechnol 59:175–181. https://doi.org/10.1016/j.copbio.2019.06.002
Guo F, Dai Z, Peng W, Zhang S, Zhou J, Ma J, Dong W, Xin F, Zhang W, Jiang M (2021) Metabolic engineering of Pichia pastoris for malic acid production from methanol. Biotechnol Bioeng 118:357–371. https://doi.org/10.1002/bit.27575
Jacobs PP, Geysens S, Vervecken W, Contreras R, Callewaert N (2009) Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology. Nat Protoc 4:58–70. https://doi.org/10.1038/nprot.2008.213
Jiang F, Doudna JA (2017) CRISPR-Cas9 structures and mechanisms annual review of biophysics. 46:505–529. https://doi.org/10.1146/annurev-biophys-062215-010822
Karbalaei M, Rezaee SA, Farsiani H (2020) Pichia pastoris: a highly successful expression system for optimal synthesis of heterologous proteins. J Cell Physiol 235:5867–5881. https://doi.org/10.1002/jcp.29583
Kiani S, Chavez A, Tuttle M, Hall RN, Chari R, Ter-Ovanesyan D, Qian J, Pruitt BW, Beal J, Vora S, Buchthal J, Kowal EJ, Ebrahimkhani MR, Collins JJ, Weiss R, Church G (2015) Cas9 gRNA engineering for genome editing, activation and repression. Nat Methods 12:1051–1054. https://doi.org/10.1038/nmeth.3580
Komor AC, Badran AH, Liu DR (2017) CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 168:20–36. https://doi.org/10.1016/j.cell.2016.10.044
Koonin EV, Makarova KS, Zhang F (2017) Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol 37:67–78. https://doi.org/10.1016/j.mib.2017.05.008
Lian J, HamediRad M, Hu S, Zhao H (2017) Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system. Nat Commun 8:1688. https://doi.org/10.1038/s41467-017-01695-x
Liao X, Zhao J, Liang S, Jin J, Li C, Xiao R, Li L, Guo M, Zhang G, Lin Y (2019) Enhancing co-translational folding of heterologous protein by deleting non-essential ribosomal proteins in Pichia pastoris. Biotechnology for biofuels 12:38. https://doi.org/10.1186/s13068-019-1377-z
Lin-Cereghino J, Hashimoto MD, Moy A, Castelo J, Orazem CC, Kuo P, Xiong S, Gandhi V, Hatae CT, Chan A, Lin-Cereghino GP (2008) Direct selection of Pichia pastoris expression strains using new G418 resistance vectors. Yeast (Chichester, England) 25:293–299 https://doi.org/10.1002/yea.1587
Lin-Cereghino J, Wong WW, Xiong S, Giang W, Luong LT, Vu J, Johnson SD, Lin-Cereghino GP (2005) Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. BioTechniques 38:44, 46, 48 https://doi.org/10.2144/05381bm04
Liu Q, Shi X, Song L, Liu H, Zhou X, Wang Q, Zhang Y, Cai M (2019) CRISPR-Cas9-mediated genomic multiloci integration in Pichia pastoris. Microbial cell factories 18:144–144. https://doi.org/10.1186/s12934-019-1194-x
Liu W, Tang D, Wang H, Lian J, Huang L, Xu Z (2019) Combined genome editing and transcriptional repression for metabolic pathway engineering in Corynebacterium glutamicum using a catalytically active Cas12a. Appl Microbiol Biotechnol 103:8911–8922. https://doi.org/10.1007/s00253-019-10118-4
Liu Y, Tu X, Xu Q, Bai C, Kong C, Liu Q, Yu J, Peng Q, Zhou X, Zhang Y, Cai M (2018) Engineered monoculture and co-culture of methylotrophic yeast for de novo production of monacolin J and lovastatin from methanol. Metab Eng 45:189–199. https://doi.org/10.1016/j.ymben.2017.12.009
Matthews CB, Wright C, Kuo A, Colant N, Westoby M, Love JC (2017) Reexamining opportunities for therapeutic protein production in eukaryotic microorganisms. Biotechnol Bioeng 114:2432–2444. https://doi.org/10.1002/bit.26378
McCarty NS, Graham AE, Studená L, Ledesma-Amaro R (2020) Multiplexed CRISPR technologies for gene editing and transcriptional regulation. Nat Commun 11:1281. https://doi.org/10.1038/s41467-020-15053-x
Näätsaari L, Mistlberger B, Ruth C, Hajek T, Hartner FS, Glieder A (2012) Deletion of the Pichia pastoris KU70 homologue facilitates platform strain generation for gene expression and synthetic biology. PLoS One 7:e39720. https://doi.org/10.1371/journal.pone.0039720
Nakamura Y, Nishi T, Noguchi R, Ito Y, Watanabe T, Nishiyama T, Aikawa S, Hasunuma T, Ishii J, Okubo Y, Kondo A (2018) A stable, autonomously replicating plasmid vector containing Pichia pastoris Centromeric DNA Applied and environmental microbiology 84:https://doi.org/10.1128/aem.02882-17
Obst U, Lu TK, Sieber V (2017) A modular toolkit for generating Pichia pastoris Secretion Libraries. ACS Synth Biol 6:1016–1025. https://doi.org/10.1021/acssynbio.6b00337
Peña DA, Gasser B, Zanghellini J, Steiger MG, Mattanovich D (2018) Metabolic engineering of Pichia pastoris. Metab Eng 50:2–15. https://doi.org/10.1016/j.ymben.2018.04.017
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 152:1173–1183
Qin X, Lu J, Zhang Y, Wu X, Qiao X, Wang Z, Chu J, Qian J (2020) Engineering Pichia pastoris to improve S-adenosyl- l-methionine production using systems metabolic strategies. Biotechnol Bioeng 117:1436–1445. https://doi.org/10.1002/bit.27300
Ramesh A, Ong T, Garcia JA, Adams J, Wheeldon I (2020) Guide RNA engineering enables dual purpose CRISPR-Cpf1 for simultaneous gene editing and gene regulation in Yarrowia lipolytica. ACS Synth Biol 9:967–971. https://doi.org/10.1021/acssynbio.9b00498
Ronda C, Maury J, Jakočiunas T, Jacobsen SA, Germann SM, Harrison SJ, Borodina I, Keasling JD, Jensen MK, Nielsen AT (2015) CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae. Microb Cell Fact 14:97. https://doi.org/10.1186/s12934-015-0288-3
Wang L, Deng A, Zhang Y, Liu S, Liang Y, Bai H, Cui D, Qiu Q, Shang X, Yang Z, He X, Wen T (2018) Efficient CRISPR-Cas9 mediated multiplex genome editing in yeasts. Biotechnol Biofuel 11:277. https://doi.org/10.1186/s13068-018-1271-0
Wang T, Guan C, Guo J, Liu B, Wu Y, Xie Z, Zhang C, Xing XH (2018) Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance. Nat Commun 9:2475. https://doi.org/10.1038/s41467-018-04899-x
Webster TD, Dickson RC (1983) Direct selection of Saccharomyces cerevisiae resistant to the antibiotic G418 following transformation with a DNA vector carrying the kanamycin-resistance gene of Tn903. Gene 26:243–252. https://doi.org/10.1016/0378-1119(83)90194-4
Weninger A, Fischer JE, Raschmanová H, Kniely C, Vogl T, Glieder A (2018) Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers. J Cell Biochem 119:3183–3198
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
Wu M, Shen Q, Yang Y, Zhang S, Qu W, Chen J, Sun H, Chen S (2013) Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115. J Ind Microbiol Biotechnol 40:589–599. https://doi.org/10.1007/s10295-013-1264-8
Xu X, Qi LS (2019) A CRISPR-dCas toolbox for genetic engineering and synthetic biology. J Mol Biol 431:34–47. https://doi.org/10.1016/j.jmb.2018.06.037
Yang Y, Liu G, Chen X, Liu M, Zhan C, Liu X, Bai Z (2020) High efficiency CRISPR/Cas9 genome editing system with an eliminable episomal sgRNA plasmid in Pichia pastoris. Enzyme Microb Technol 138:109556. https://doi.org/10.1016/j.enzmictec.2020.109556
Yang Z, Zhang Z (2018) Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: a review. Biotechnol Adv 36:182–195. https://doi.org/10.1016/j.biotechadv.2017.11.002
Zhang Y, Wang J, Wang Z, Zhang Y, Shi S, Nielsen J, Liu Z (2019) A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nat Commun 10:1053. https://doi.org/10.1038/s41467-019-09005-3
Funding
This work was supported by the National Key Research and Development Program of China (Grant No. 2018YFA0901500) and the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project TSBICIP-KJGG-006.
Author information
Authors and Affiliations
Contributions
XL and CZ conceived and designed research. XL and LL conducted experiments and analyzed data. XL wrote the manuscript. CZ, AJ, and XX revised the manuscript. All authors read and approved the manuscript.
Corresponding author
Ethics declarations
Ethics approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Liao, X., Li, L., Jameel, A. et al. A versatile toolbox for CRISPR-based genome engineering in Pichia pastoris. Appl Microbiol Biotechnol 105, 9211–9218 (2021). https://doi.org/10.1007/s00253-021-11688-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-021-11688-y