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

• Published in: FEMS yeast research Vol. 24
• Main Authors: Strucko, Tomas, Gadar-Lopez, Adrian-E, Frøhling, Frederik B, Frost, Emma T, Iversen, Esther F, Olsson, Helen, Jarczynska, Zofia D, Mortensen, Uffe H
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 09.01.2024
• Subjects: CRISPR-Cas Systems; DNA End-Joining Repair; Gene Deletion; Gene Editing - methods; Genetic Engineering - methods; Genetic Vectors - genetics; Oligonucleotides - genetics; Saccharomycetales - genetics; KU70; transient-NHEJ; multiplexing; uidA marker; Cas9; mutagenesis; oligonucleotide
• ISSN: 1567-1364; 1567-1364
• DOI: 10.1093/femsyr/foae026

Abstract 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 (NHEJ) 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. A streamlined CRISPR-Cas9-based toolbox that improves the engineering process of Komagataella phaffii yeast by implementing short single-stranded DNA sequences for precise introduction of desired genetic modifications.