Genetic drivers of Chinese hamster ovary cell proliferation revealed by functional genomics.

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.