Functional genomic screening in Komagataella phaffii enabled by high-activity CRISPR-Cas9 library

Area of Science:

• * Molecular Biology
• * Yeast Genetics
• * Functional Genomics

Background:

• * The yeast Komagataella phaffii is a key host in the biopharmaceutical industry and metabolic engineering.
• * High-throughput genome-wide loss-of-function screens using CRISPR are essential for functional genetics and strain engineering.
• * Efficient tools are needed to understand K. phaffii's genetic landscape for optimizing industrial applications.

Purpose of the Study:

• * To design and validate a high-activity, genome-wide sgRNA library for K. phaffii.
• * To enable precise functional genomic screening in K. phaffii.
• * To identify essential genes in K. phaffii, particularly those relevant to metabolic engineering.

Main Methods:

• * Development of a 6-fold coverage genome-wide sgRNA library (30,848 active sgRNAs) targeting over 99% of K. phaffii coding sequences.
• * Conducting fitness screens in K. phaffii lacking functional non-homologous end joining (NHEJ) to validate sgRNA activity.
• * Performing growth screens using glucose as the sole carbon source to identify essential genes.

Main Results:

• * A highly active and comprehensive genome-wide CRISPR library for K. phaffii was successfully designed and validated.
• * Experimental validation of sgRNA targeting activity was achieved by screening in the absence of NHEJ.
• * A core set of essential genes in K. phaffii was identified, including genes involved in protein production, secretion, and glycosylation.

Conclusions:

• * The developed CRISPR library significantly enhances functional genomic screening capabilities in K. phaffii.
• * The identified essential genes provide valuable targets for optimizing K. phaffii for biopharmaceutical and metabolic engineering purposes.
• * This resource facilitates future genetic screens and strain engineering efforts in this industrially relevant yeast.