A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9 | A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9

Background The yeast Komagataella phafi (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. phafi 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 efciency and suitability for producing secreted recombinant food proteins. Results Three Cas9/sgRNA plasmids were constructed, along with corresponding donor helper plasmids con‑ taining homology regions for chromosomal integration via homology-directed repair. The integration efciency of an enhanced green fuorescent protein (eGFP) expression cassette was assessed at three genomic loci (04576, PFK1, and ROX1). The 04576 locus showed the highest integration efciency, while ROX1 had the highest transformation efciency. Whole genome sequencing revealed variable copy numbers of eGFP expression cassettes among clones, corresponding with increasing levels of fuorescence. Furthermore, the system’s applicability for producing recombi‑ nant food proteins was validated by successfully expressing and secreting chicken ovalbumin. This constitutes the frst report of CRISPR/Cas9 applied to produce recombinant chicken ovalbumin. Conclusions The adapted GoldenPiCS toolkit combined with CRISPR/Cas9 technology enabled efcient and precise genome integration in K. phafi. This approach holds promise for expanding the production of high-value recom‑ binant proteins. Future research should focus on optimizing integration sites and improving cloning procedures to enhance the system’s efciency and versatility. Keywords Pichia pastoris, Komagataella phafi, CRISPR/Cas9, Heterologous expression, Golden Gate assembly, Food proteins, Ovalbumin, WGS

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