• Double knockout of OAZ1 and CASP8AP2 in HEK293 cells significantly reduces apoptosis rates, enhancing cell viability during culture.
• Recombinant protein yields for SEAP and vitronectin increase by 2.1-fold and 2.9-fold, respectively, compared to wild-type cells.
• Metabolic reprogramming is evidenced by G1/G0 cell cycle arrest and altered consumption/production rates of key metabolites.
• This dual-target CRISPR/Cas9 strategy offers a novel approach to improve HEK293-based biopharmaceutical production.