Treffer: Engineering Pichia pastoris Strains Using CRISPR/Cas9 Technologies: The Basic Protocol.

Title:
Engineering Pichia pastoris Strains Using CRISPR/Cas9 Technologies: The Basic Protocol.
Authors:
Raschmanová H; University of Chemistry and Technology Prague, Department of Biotechnology, Prague, Czech Republic., Weninger A; Graz University of Technology, Institute of Molecular Biotechnology, Graz, Austria., Kovar K; daspool, Wädenswil, Switzerland.
Source:
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2026; Vol. 2697, pp. 361-371.
Publication Type:
Journal Article
Language:
English
Journal Info:
Publisher: Humana Press Country of Publication: United States NLM ID: 9214969 Publication Model: Print Cited Medium: Internet ISSN: 1940-6029 (Electronic) Linking ISSN: 10643745 NLM ISO Abbreviation: Methods Mol Biol Subsets: MEDLINE
Imprint Name(s):
Publication: Totowa, NJ : Humana Press
Original Publication: Clifton, N.J. : Humana Press,
MeSH Terms:
CRISPR-Cas Systems*/genetics , Gene Editing*/methods , Saccharomycetales*/genetics , Metabolic Engineering*/methods , Pichia*/genetics, RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Fungal
References:
Mojica FJM, Díez-Villaseñor C, García-Martínez J, Soria E (2005) Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 60:174–182. https://doi.org/10.1007/s00239-004-0046-3. (PMID: 10.1007/s00239-004-0046-315791728)
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. https://doi.org/10.1126/science.1231143. (PMID: 10.1126/science.1231143232877183795411)
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 337:816–821. https://doi.org/10.1126/science.1225829. (PMID: 10.1126/science.1225829227452496286148)
Raschmanová H, Weninger A, Glieder A, Kovar K, Vogl T (2018) Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: current state and future prospects. Biotechnol Adv 36:641–665. https://doi.org/10.1016/j.biotechadv.2018.01.006. (PMID: 10.1016/j.biotechadv.2018.01.00629331410)
Weninger A, Hatzl AM, Schmid C, Vogl T, Glieder A (2015) 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. (PMID: 10.1016/j.jbiotec.2016.03.027)
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. https://doi.org/10.1002/jcb.26474. (PMID: 10.1002/jcb.2647429091307)
Fischer JE, Glieder A (2022) CRISPR/Cas9 tool kit for efficient and targeted insertion/deletion mutagenesis of the Komagataella phaffii (Pichia pastoris). Genome Methods Mol Biol 2513:121–133. https://doi.org/10.1007/978-1-0716-2399-2_8. (PMID: 10.1007/978-1-0716-2399-2_835781203)
Contributed Indexing:
Keywords: CRISPR/Cas9; Gene knockout; Genome editing; Genome engineering; Guide RNA; Homologous recombination; Komagataella phaffii; Pichia pastoris; Synthetic biology
Substance Nomenclature:
0 (RNA, Guide, CRISPR-Cas Systems)
SCR Organism:
Komagataella pastoris
Entry Date(s):
Date Created: 20251001 Date Completed: 20251001 Latest Revision: 20251001
Update Code:
20251001
DOI:
10.1007/978-1-0716-4779-0_25
PMID:
41028476
Database:
MEDLINE

Weitere Informationen

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) system has become a commonly used tool for genome editing and metabolic engineering. For Komagataella phaffii, commercialized as Pichia pastoris, the CRISPR/Cas9 protocol for genome editing was established in 2016 and since then has been employed to facilitate genetic modifications such as markerless gene disruptions and deletions as well as to enhance the efficiency of homologous recombination.In this chapter, we describe a robust basic protocol for CRISPR-based genome editing, demonstrating near 100% targeting efficiency for gene inactivation via a frameshift mutation. As described in other chapters of this volume, CRISPR/Cas9 technologies for use in P. pastoris have been further optimized for various specific purposes.
(© 2026. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)