A high-throughput co-culture CRISPR array for rapid fitness hypothesis testing and growth engineering in CHO cells

Cell growth and fitness shape mammalian biomanufacturing performance, but subtle genetic effects are difficult to resolve with conventional growth assays. We developed a high-throughput competitive co-culture CRISPR array in Chinese hamster ovary (CHO) suspension cells that pairs a CRISPR-competent population with a CRISPR-inactive green fluorescent protein (GFP) reference pool and tracks GFP-ratio dynamics by flow cytometry. Guided by a prior genome-wide CRISPR screen, we tested 235 single-gene knockouts and 155 multiplex gene sets across seven batches and >100 days of cultivation. The assay detected expected lethal and growth-inhibitory perturbations and identified 28 growth-enhancing knockouts or gene sets that are poorly resolved by viable-cell-concentration measurements. Slope-based modeling converted GFP dynamics into inferred growth rate changes, and combinatorial exclusion with bulk transcriptomics linked multiplex phenotypes to candidate driver genes. The approach enables scalable fitness hypothesis testing and prioritization of CHO growth-engineering targets for downstream host-cell development.