ArticleJournal of cellular biochemistry2018
Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers.
Article in Journal of cellular biochemistry, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 66 papers, 1 of them a synthesis that pooled it.
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Who cites it
66 citing papers in PubMed, 1 synthesis or guideline pooled it, 124 citations in OpenAlex.
- A Systematic Review of the Potential of Pichia pastoris (Komagataella phaffii) as an Alternative Host for Biologics Production.Molecular biotechnology · 2024Pooled it
- Yeast species resource exploration and application in cell factories.Synthetic and systems biotechnology · 2026Review
- An advanced genome engineering platform for Komagataella phaffii enabling versatile in vivo DNA assembly and multiplex integration.Journal of biological engineering · 2026Article
- Systematic metabolic engineering of Komagataella phaffii for methanol-to-lactate bioconversion.Bioprocess and biosystems engineering · 2026Article
- A CRISPR/Cas9-based genome-editing platform enabling efficient and precise gene replacement in Lipomyces starkeyi.FEMS yeast research · 2026Article
- Adaptive Laboratory Evolution of Pichia pastoris by Serial Batch Cultivation.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Engineering small peptide secretion inFrontiers in bioengineering and biotechnology · 2026Article
- Engineering Pichia pastoris Strains Using CRISPR/Cas9 Technologies: The Basic Protocol.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Auxin-Induced Degradation of Target Proteins in Pichia pastoris.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Promoter engineering for enhanced 3-hydroxypropionic acid production inSynthetic and systems biotechnology · 2025Article
- Sterol regulation in Komagataella phaffii: Identification of sterol regulatory element binding proteins and their activation pathway.The Journal of biological chemistry · 2025Article
- A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9.Microbial cell factories · 2025Article
- Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha.Microbial cell factories · 2025Article
- Applications of the Methylotrophic YeastJournal of fungi (Basel, Switzerland) · 2024Review
- A CRISPR/Cas9-based visual toolkit enabling multiplex integration at specific genomic loci inSynthetic and systems biotechnology · 2024Article
- Identification of a novel growth-associated promoter for biphasic expression of heterogenous proteins inApplied and environmental microbiology · 2024Article
- CRISPR/Cas9-based toolkit for rapid marker recycling and combinatorial libraries in Komagataella phaffii.Applied microbiology and biotechnology · 2024Article
- Human Sterols Are Overproduced, Stored and Excreted in Yeasts.International journal of molecular sciences · 2024Article
- Multiplex Marker-Less Genome Integration in Pichia pastoris Using CRISPR/Cas9.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Enhanced Expression of Alcohol Dehydrogenase I inMicroorganisms · 2023Article
6 more citing papers are in PubMed but not listed here.
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
6 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non-homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ∼20% correct integration using CRISPR/Cas9. Here, we demonstrate the CRISPR/Cas9 mediated integration of markerless donor cassettes at efficiencies approaching 100% using a ku70 deletion strain. The Ku70p is involved in NHEJ repair and lack of the protein appears to favor repair via HR near exclusively. While the absolute number of transformants in the Δku70 strain is reduced, virtually all surviving transformants showed correct integration. In the wildtype strain, markerless donor cassette integration was also improved up to 25-fold by placing an autonomously replicating sequence (ARS) on the donor cassette. Alternative strategies for improving donor cassette integration using a Cas9 nickase variant or reducing off targeting associated toxicity using a high fidelity Cas9 variant were so far not successful in our hands in P. pastoris. Furthermore we provide Cas9/gRNA expression plasmids with a Geneticin resistance marker which proved to be versatile tools for marker recycling. The reported CRSIPR-Cas9 tools can be applied for modifying existing production strains and also pave the way for markerless whole genome modification studies in P. pastoris.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.