Utilizing Stable Gene-Edited Knockout Pools for Genetic Screening and Engineering in Chinese Hamster Ovary Cells.

Chinese hamster ovary (CHO) cells are the primary host for biopharmaceutical production. To meet increasing demands for productivity, quality, and complex molecule expression, genetic engineering, particularly clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene knockout (KO), is widely used to optimize host cell performance. However, systematic screening of KO targets remains challenging due to the labor-intensive process of generating and evaluating individual clones. In this study, we present a robust, high-throughput CRISPR workflow using stable KO pools in CHO cells. These pools maintain genetic stability for over 6 weeks, including in multiplexed configurations targeting up to seven genes simultaneously. Compared to clonal approaches, KO pools reduce variability caused by clonal heterogeneity and better reflect the host cell population phenotype. We demonstrate the utility of this approach by reproducing the beneficial phenotypic effects of fibronectin 1 (FN1) KO, specifically prolonged culture duration and improved late-stage viability in fed-batch processes. This workflow enables efficient identification and evaluation of promising KO targets without the need to generate and test large numbers of clones. Overall, screening throughput is increased 2.5-fold and timelines are compressed from 9 to 5 weeks. This provides a scalable, efficient alternative to traditional clonal screening, accelerating discovery for CHO cell line engineering for biopharmaceutical development.