Abstract
Cell 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 biopharmaceutical production cell lines. To uncover genetic drivers of proliferation in Chinese hamster ovary (CHO) cells, we performed a genome-wide CRISPR knockout-screen targeting 17,761 expressed genes. Using a stable Cas9-expressing CHO pool, low-multiplicity of infection (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. We identified 235 putative 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 CRISPR screening framework and a genetic blueprint for engineering faster-growing CHO cell lines to support accelerated cell line development.