CASP8AP2 Knockout Cell Line (HEK293)

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.

Human embryonic kidney (HEK) 293 cells are widely used for recombinant protein production because of their efficient posttranslational modification capabilities. However, their large-scale culture is often limited by metabolic stress and early apoptosis, leading to insufficient protein yields. In this study, we aim to increase protein expression through the coordinated modulation of metabolic and apoptotic pathways. Using CRISPR/Cas9 technology, we target and knockout the genes of ornithine decarboxylase antizyme 1 (OAZ1), which regulates polyamine metabolism, and caspase 8-associated protein 2 (CASP8AP2), an apoptosis-related protein. We successfully construct an / double-knockout HEK293 cell line. Following transfection with the knockout vector and screening of single-cell clones, multiple levels of validation confirm the successful gene knockout. The results show that the double-knockout cells exhibit significantly reduced apoptosis rates. Furthermore, the production of recombinant secreted alkaline phosphatase (SEAP) and vitronectin (VN) increases by 2.1 folds and 2.9 folds, respectively, compared with those in wild-type cells. Metabolic profiling reveals that the cell cycle is arrested in the G1/G0 phase, accompanied by increased specific consumption and production rates of key metabolites. This study demonstrates that concurrent inhibition of apoptosis and optimization of metabolism effectively enhances recombinant protein production in HEK293 cells, suggesting a novel strategy for improving HEK293 cell-based expression.