Knockout of the caspase 8‐associated protein 2 gene improves recombinant protein expression in HEK293 cells through up‐regulation of the cyclin‐dependent kinase inhibitor 2A gene

Cell lines used in bioproduction are routinely engineered to improve their production efficiency. Numerous strategies, such as random mutagenesis, RNA interference screens, and transcriptome analyses have been employed to identify effective engineering targets. A genome‐wide small interfering RNA screen previously identified the CASP8AP2 gene as a potential engineering target for improved expression of recombinant protein in the HEK293 cell line. Here, we validate the CASP8AP2 gene as an engineering target in HEK293 cells by knocking it out using CRISPR/Cas9 genome editing and assessing the effect of its knockout on recombinant protein expression, cell growth, cell viability, and overall gene expression. HEK293 cells lacking CASP8AP2 showed a seven‐fold increase in specific expression of recombinant luciferase and a 2.5‐fold increase in specific expression of recombinant SEAP, without significantly affecting cell growth and viability. Transcriptome analysis revealed that the deregulation of the cell cycle, specifically the upregulation of the cyclin‐dependent kinase inhibitor 2A (CDKN2A) gene, contributed to the improvement in recombinant protein expression in CASP8AP2 deficient cells. The results validate the CASP8AP2 gene is a viable engineering target for improved recombinant protein expression in the HEK293 cell line. Knocking out Caspase 8 associated protein 2 (CASP8AP2) through siRNA screening was associated with improved expression of recombinant protein without affecting cell growth and viability. Validation of this finding by knockout CASP8AP2 using CRISPR/cas9 genome editing resulted in 7‐fold increase in luciferase expression. Cell cycle and whole transcriptome analyses revealed that the increased luciferase expression was due to G0/G1 cell cycle arrest orchestrated by upregulated cyclin dependent kinase inhibitor 2A (CDKN2A) gene expression in the CASP8AP2 knockout cells.