High-Throughput Generation of Pichia pastoris Knock-Out Strains by Using CRISPR/Cas9

Area of Science:

• Molecular Biology
• Microbial Genetics
• Biotechnology

Background:

• The CRISPR/Cas9 system is a revolutionary tool for genome engineering, expanding its application across various organisms.
• Previous CRISPR/Cas9 tools for Komagataella phaffii allowed mutant strain generation in under two weeks.
• Existing K. phaffii CRISPR systems utilize episomal vectors, enabling plasmid and marker recycling for clean mutant strains.

Purpose of the Study:

• To describe a high-throughput pipeline for generating Komagataella phaffii mutant strains using CRISPR/Cas9.
• To detail a four-step process for creating gene-interrupted K. phaffii mutants via NHEJ repair.
• To facilitate reproducible and simplified research through a 96-well format compatible procedure.

Main Methods:

• CRISPR/Cas9 plasmids assembly.
• Transformation of Komagataella phaffii.
• Screening for mutant strains and plasmid elimination.

Main Results:

• A reproducible four-step pipeline for high-throughput generation of K. phaffii mutant strains was established.
• The method allows for the generation of mutant strains with interrupted open reading frames.
• The system utilizes episomal vectors for CRISPR/Cas9 expression, facilitating plasmid elimination and resulting in clean mutant strains.

Conclusions:

• The developed CRISPR/Cas9 pipeline offers an efficient and high-throughput method for Komagataella phaffii genome engineering.
• The protocol's reproducibility and adaptability to a 96-well format simplify and accelerate research.
• The ability to obtain empty mutant strains makes this system ideal for subsequent genetic investigations.