ENAGenomicsSSCSartorius Stedim Cellca GmbHhttps://www.ebi.ac.uk/ena/browser/view/PRJEB91812Cell lines used in biopharmaceutical manufacturing are engineered to optimize protein yield, quality, and production speed. Cellular growth rate is a key bottleneck in the development of high-yield biopharmaceutical production cell lines. To systematically uncover genetic drivers of proliferation in Chinese hamster ovary (CHO) cells, we performed a high-coverage, genome-wide CRISPR knockout screen targeting 17 761 expressed genes with six sgRNAs per gene. Using a stable Cas9-expressing CHO pool, low-MOI lentiviral transduction, and cultivation under both shake-flask and 3 L perfusion bioprocess conditions, we maintained >5 000× library coverage with predominantly single-copy integrations. Differential analysis across multiple timepoints identified 235 growth-enhancing gene knockouts, clustered into 155 functional gene sets, spanning pathways related to chromatin regulation, cell cycle progression, and nutrient sensing. In parallel, we identified a set of 3 110 genes essential for cell fitness (CHO_EG2025), providing a resource for CHO functional genomics. This work establishes a scalable CRISPR screening framework and a comprehensive genetic blueprint for engineering faster-growing CHO cell lines to support accelerated cell line development and biopharmaceutical manufacturing.ENAfalseGenetic Drivers of Chinese Hamster Ovary Cell Proliferation Revealed by Functional GenomicsCell lines used in biopharmaceutical manufacturing are engineered to optimize protein yield, quality, and production speed. Cellular growth rate is a key bottleneck in the development of high-yield biopharmaceutical production cell lines. To systematically uncover genetic drivers of proliferation in Chinese hamster ovary (CHO) cells, we performed a high-coverage, genome-wide CRISPR knockout screen targeting 17 761 expressed genes with six sgRNAs per gene. Using a stable Cas9-expressing CHO pool, low-MOI lentiviral transduction, and cultivation under both shake-flask and 3 L perfusion bioprocess conditions, we maintained >5 000× library coverage with predominantly single-copy integrations. Differential analysis across multiple timepoints identified 235 growth-enhancing gene knockouts, clustered into 155 functional gene sets, spanning pathways related to chromatin regulation, cell cycle progression, and nutrient sensing. In parallel, we identified a set of 3 110 genes essential for cell fitness (CHO_EG2025), providing a resource for CHO functional genomics. This work establishes a scalable CRISPR screening framework and a comprehensive genetic blueprint for engineering faster-growing CHO cell lines to support accelerated cell line development and biopharmaceutical manufacturing.2025-08-312025-08-31PRJEB91812