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

High-throughput genome-wide loss-of-function screens using CRISPR-Cas9 technology have emerged as a novel approach in metabolic engineering offering insight into cell biology and strain engineering. Komagataella phaffii is a yeast host of particular interest in the biopharmaceutical industry. Here we successfully designed and validated 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 expressed genes. By quantifying the targeting efficiency if each of the guides in the library, we improved the precision of essential gene identification under glucose-supported growth condition. In addition, comparative analysis of essential genes characterized in this screen and those found in other yeast species enable the definition of a set of core essential genes crucial for vital cell processes, as well as identification of a unique set of essential genes exclusive to K. phaffii. This contributes to an enhanced understanding of this cell's biology and offers a novel tool for further genetic modification of this host.