Metabolic engineeringJul 17, 2024

Using a powerful CRISPR-Cas9 tool to study gene functions in Komagataella phaffii

Updated Apr 1, 2026

Abstract

A genome-wide sgRNA library containing 30,848 active sgRNAs targeting over 99% of coding sequences has been developed for the yeast Komagataella phaffii.

This library allows for improved functional genetic screening and strain engineering in K. phaffii.
Fitness screens conducted in the absence of non-homologous end joining (NHEJ) enable quantitative assessment of sgRNA activity.
Growth screens identified essential genes in K. phaffii when using glucose as the sole carbon source.
A core set of essential genes was identified, many of which are linked to metabolic engineering targets such as protein production and secretion.
The developed CRISPR library may facilitate future genetic screens beyond gene essentiality classification.

Simplified

CRISPR-based high-throughput genome-wide loss-of-function screens are a valuable approach to functional genetics and strain engineering. The yeast Komagataella phaffii is a host of particular interest in the biopharmaceutical industry and as a metabolic engineering host for proteins and metabolites. Here, we design and validate a highly active 6-fold coverage genome-wide sgRNA library for this biotechnologically important yeast containing 30,848 active sgRNAs targeting over 99% of its coding sequences. Conducting fitness screens in the absence of functional non-homologous end joining (NHEJ), the dominant DNA repair mechanism in K. phaffii, provides a quantitative means to assess the activity of each sgRNA in the library. This approach allows for the experimental validation of each guide's targeting activity, leading to more precise screening outcomes. We used this approach to conduct growth screens with glucose as the sole carbon source and identify essential genes. Comparative analysis of the called gene sets identified a core set of K. phaffii essential genes, many of which relate to metabolic engineering targets, including protein production, secretion, and glycosylation. The high activity, genome-wide CRISPR library developed here enables functional genomic screening in K. phaffii, applied here to gene essentiality classification, and promises to enable other genetic screens.

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