ArticleMicrobial cell factories2025
A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9.
Article in Microbial cell factories, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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Who cites it
3 citing papers in PubMed.
- Metabolic engineering ofApplied and environmental microbiology · 2026Review
- CRISPR-enhanced microalgae biosynthesis: a promising approach for future functional feed ingredients.Journal of animal science and biotechnology · 2026Review
- Toward food-grade production of the Glutamicibacter halophytocola diamine oxidase using Komagataella phaffii.AMB Express · 2025Article
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5 authors.
Funding
Abstract
backgroundThe yeast Komagataella phaffii (formerly known as Pichia pastoris) has been widely used for functional expression of recombinant proteins, including plant and animal food proteins. CRISPR/Cas9 genome editing systems can be used for insertion of heterologous genes without the use of selection markers. The study aimed to create a convenient markerless knock-in method for integrating expression cassettes into the chromosome of K. phaffii using CRISPR/Cas9 technology. The approach was based on the hierarchical, modular, Golden Gate assembly employing the GoldenPiCS toolkit. Furthermore, the aim was to evaluate the system's efficiency and suitability for producing secreted recombinant food proteins.
resultsThree Cas9/sgRNA plasmids were constructed, along with corresponding donor helper plasmids containing homology regions for chromosomal integration via homology-directed repair. The integration efficiency of an enhanced green fluorescent protein (eGFP) expression cassette was assessed at three genomic loci (04576, PFK1, and ROX1). The 04576 locus showed the highest integration efficiency, while ROX1 had the highest transformation efficiency. Whole genome sequencing revealed variable copy numbers of eGFP expression cassettes among clones, corresponding with increasing levels of fluorescence. Furthermore, the system's applicability for producing recombinant food proteins was validated by successfully expressing and secreting chicken ovalbumin. This constitutes the first report of CRISPR/Cas9 applied to produce recombinant chicken ovalbumin.
conclusionsThe adapted GoldenPiCS toolkit combined with CRISPR/Cas9 technology enabled efficient and precise genome integration in K. phaffii. This approach holds promise for expanding the production of high-value recombinant proteins. Future research should focus on optimizing integration sites and improving cloning procedures to enhance the system's efficiency and versatility.
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