• Double knockout of OAZ1 and CASP8AP2 in HEK293 cells significantly reduces apoptosis rates.
• Recombinant protein production of SEAP and VN increases by 2.1-fold and 2.9-fold, respectively, compared to wild-type cells.
• Metabolic reprogramming is evidenced by G1/G0 phase cell cycle arrest and altered metabolite consumption/production rates.
• Concurrent modulation of apoptosis and metabolism via CRISPR/Cas9 provides a novel strategy to enhance recombinant protein yields in HEK293 cells.