Same hydrolase knockouts, different outcomes: CRISPR/Cas9 editing workflow shapes resulting cellular phenotype and transcriptome in CHO

CRISPR/Cas9-based multi-gene knockout strategies are widely applied to engineer Chinese hamster ovary (CHO) cells, yet the impact of editing workflow on cellular outcomes remains poorly understood. Here, we systematically compare sequential and multiplex genome editing approaches to generate CHO cell lines lacking nine polysorbate (PS)-degrading hydrolases. Both strategies achieved comparable functional outcomes, markedly improving PS stability. However, they produced distinct phenotypic and transcriptomic trajectories. Sequential editing led to progressive declines in growth and viability, increased cell size and DNA content, and extensive transcriptomic remodeling. In contrast, multiplex editing resulted in milder phenotypic alterations and a substantially smaller transcriptional footprint, albeit with increased clone-to-clone heterogeneity. Transcriptome analyses revealed limited overlap between final states, indicating workflow-specific adaptation despite the same intended knockout set. Importantly, transcriptional changes were primarily associated with clonal selection and iterative manipulation rather than reflecting a shared response to the targeted knockouts themselves. These findings demonstrate that genome editing workflows can shape cellular phenotypes independently of intended genetic modifications. For cell line development, multiplex editing offers a strategy to reduce development time and limit undesirable drift when targeting non-essential genes, highlighting the need to consider engineering strategy as a critical design parameter.