A Novel Chassis for High-quality Recombinant Protein Expression: Apoptosis Pathway Engineering via Multi-gene Knockout and Efficacy Improvement of CHO Cells

Background Chinese hamster ovary (CHO) cells are pivotal in biopharmaceutical production, yet balancing high recombinant protein yield with cell survival remains challenging. While previous studies have targeted single apoptotic regulators, the synergistic effects of multi-gene ablation on protein stability are unknown. Results This study presents a multi-knockout strategy for CHO cells. Herewithin, CHO-K1 knockout lines were engineered via CRISPR-Cas9 targeting of key triple (TKO, BAK/BAX/CASP3), double (DKO, BAK/BAX), and single (SKO, CASP3) apoptotic nodes. Subsequently, comprehensive analyses of apoptosis, cell viability, doubling time, cell cycle and mitochondrial membrane potential were conducted for isolated clones. The triple knockout cell lines exhibited the highest overall levels of cell viability, a prolonged doubling time, and enhanced resistance to apoptosis. These characteristics directly translated to improved expression of recombinant blue fluorescent protein, with triple knockouts outperforming WT and single/double knockout lines. Conclusions These findings establish a robust foundation for engineering apoptosis-resistant CHO cell lines with enhanced protein production capacity, offering a promising approach for improved efficiency and reduced costs in biopharmaceutical manufacturing.