Paving the Way for a Minimal CHO ChassisExploring the Upper Limits of Megabase-Scale Deletions through DNA Repair Modulation.

Chinese hamster ovary (CHO) cells are the predominant mammalian host for therapeutic protein production, yet their large genome contains substantial non-essential DNA that may impose metabolic burden and contribute to process variability. Here, we establish a CRISPR/Cas9-based strategy for megabase-scale genomic deletions in CHO cells, targeting regions from 3 to over 100 megabases (Mb). Clones carrying deletions of up to 50 Mb were isolated, while a candidate deletion junction spanning 130 Mb was detected in the edited pool, demonstrating the potential for large-scale genome engineering in mammalian cells. Importantly, removal of genomic regions with minimal coding content did not impair growth, viability, or monoclonal antibody productivity. Mechanistic analysis revealed rapid deletion formation and a size-dependent shift in DNA repair pathway usage. Transient inhibition of non-homologous end joining (NHEJ) significantly improved deletion efficiency and clone recovery without compromising cell fitness. These findings establish large-scale genome reduction as a feasible and controllable strategy for rational CHO genome streamlining and the development of next-generation biopharmaceutical cell factories.