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

Oct 1, 2025Methods in molecular biology (Clifton, N.J.)

Easy Creation of Pichia pastoris Gene Knock-Outs with CRISPR

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Abstract

CRISPR/Cas9 tools can generate mutant strains of Komagataella phaffii in less than 2 weeks.

The CRISPR/Cas9 system has been adapted for use in Komagataella phaffii, expanding its applicability in genome engineering.
Episomal vectors are utilized for expressing CRISPR/Cas9 elements, allowing for the recycling of plasmids after editing.
Engineered strains do not retain the resistance marker or CRISPR/Cas9 plasmid in their genome, facilitating further studies.
A high-throughput pipeline for generating mutant strains involves four steps: plasmid assembly, transformation, screening, and plasmid elimination.
Most procedures in the pipeline can be performed in a 96-well format, enhancing reproducibility and efficiency.

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The CRISPR/Cas9 (CRISPR is an acronym for clustered regularly interspaced short palindromic repeats) system is a powerful molecular biological tool simplifying the process of genome engineering. Originally demonstrated to function in human and mouse cells, the portfolio of organisms that can be engineered by the new and groundbreaking technology was soon expanded. In the past years, CRISPR/Cas9 tools for use in Komagataella phaffii were reported to allow the generation of K. phaffii mutant strains in less than 2 weeks. In addition, the K. phaffii tailored system uses episomal vectors for the expression of the CRISPR/Cas9 elements, which allows the recycling of the plasmid after the CRISPR editing to obtain empty mutant strains. This means that the engineered strains do not carry the expression cassette of the resistance marker and CRISPR/Cas9 plasmid in their genome and are therefore a superb starting point for further investigations.In this chapter, we describe a pipeline for the high-throughput generation of K. phaffii mutant strains with interrupted open reading frames of genes, by using the CRISPR/Cas9 system in combination with error-prone repair of the double-strand break by NHEJ. The pipeline we developed consists of four steps: (a) CRISPR/Cas9 plasmids assembly, (b) transformation of K. phaffii, (c) screening for mutant strains, and (d) plasmid elimination and is due to the detailed description of every step being easily reproducible. To intensify and simplify the research work, most of the described procedures can be performed in a 96-well format.

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