Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering of Komagataella phaffii

Aug 23, 2024FEMS yeast research

Using short DNA tools with CRISPR-Cas9 for efficient genetic editing of Komagataella phaffii

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Abstract

A versatile CRISPR-Cas9 method was developed for efficient gene editing in Komagataella phaffii.

Gene editing was achieved using linear DNA fragments with short targeting sequences, including single-stranded oligonucleotides.
Site-specific point mutations and full gene deletions were performed with high efficiency using 90 nt single-stranded oligonucleotides.
A strategy was designed to transiently inactivate the nonhomologous end-joining pathway by disrupting the KU70 gene with a visual marker.
This system allows for precise CRISPR-Cas9 editing and the possibility of multiplexing.
The tools developed can facilitate easy and efficient engineering of K. phaffii strains and are compatible with high-throughput workflows.

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Komagataella phaffii (Pichia pastoris) is a methylotrophic yeast that is favored by industry and academia mainly for expression of heterologous proteins. However, its full potential as a host for bioproduction of valuable compounds cannot be fully exploited as genetic tools are lagging behind those that are available for baker's yeast. The emergence of CRISPR-Cas9 technology has significantly improved the efficiency of gene manipulations of K. phaffii, but improvements in gene-editing methods are desirable to further accelerate engineering of this yeast. In this study, we have developed a versatile vector-based CRISPR-Cas9 method and showed that it works efficiently at different genetic loci using linear DNA fragments with very short targeting sequences including single-stranded oligonucleotides. Notably, we performed site-specific point mutations and full gene deletions using short (90 nt) single-stranded oligonucleotides at very high efficiencies. Lastly, we present a strategy for transient inactivation of nonhomologous end-joining () pathway, where KU70 gene is disrupted by a visual marker (uidA gene). This system enables precise CRISPR-Cas9-based editing (including multiplexing) and facilitates simple reversion to NHEJ-proficient genotype. In conclusion, the tools presented in this study can be applied for easy and efficient engineering of K. phaffii strains and are compatible with high-throughput automated workflows.

Key numbers

90%

Gene Targeting Efficiency

Percentage of transformants that were fluorescent, indicating successful gene integration.

75%

Transformation Success Rate

Success rate of gene deletions using a 90-nt oligonucleotide in CRISPR experiments.

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Abstract

Key numbers

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