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

Abstract

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. K E Y W O R D S caspase 8-associated protein 2, CRISPR/Cas9, cyclin-dependent kinase inhibitor 2A, recombinant protein, RNA-seq analysis 1 | INTRODUCTION Improved expression of recombinant proteins from mammalian cells is the desired goal being pursued through many different strategies. The main focus has been on optimizing growth and culture parameters (De Jesus & Wurm, 2011) and on modifying the properties of the producing cells (Fischer et al., 2015). The gene targets for cell modification are usually selected based on their known biological functions and the desired phenotypic outcome. For example, proapoptotic and antiapoptotic genes have been manipulated to extend the longevity of cultured cells (Baek et al., 2017; Cost et al., 2010); and genes involved in sugar transport have been manipulated to improve the metabolic efficiency of the producing cells (Inoue et al., 2010; Paredes et al., 1999). However, this approach is limited to genes with known function, whose altered expression may achieve the desired phenotype. In addition, genetic redundancy or other system-level robustness can nullify attempted modifications (Ekholm & Reed, 2000). As a result, novel approaches for identifying engineering targets are needed.

Knockout of caspase-7 gene improves the expression of recombinant protein in CHO cell line through the cell cycle arrest in G2/M phase

“…The higher JRed expression (1.5-fold) in CHO-KO cells compared to CHO-K1 cells also confirmed the primary findings. In line with this evidence, Abaandou et al reported that the caspase 8‐associated protein 2 (CASP8AP2) gene silencing improved recombinant protein expression in HEK293 cells by inducing cell cycle arrest in G0/G1 checkpoint [ 34 ]. They demonstrated that silencing CASP8AP2 lead to the enhancement of luciferase and SEAP production by up to 7- and 2.5-fold respectively.…”

“…CHO-KO and native CHO-K1 cells were plated in 96-well plates with a concentration of 5 × 10 3 per well in quintuplicate. Cells were incubated for 24, 48, 72 and 96 h [ 34 ] and after that doubling time of cells was identified by using cell counter. For cell viability assay the medium from each well was entirely removed and replaced with 100 µL of fresh serum-free culture medium containing 5 mg/mL MTT solution.…”

“…CHO-KO and native CHO-K1 cells were seeded in the 6-well plate by the concentration of 5 × 10 5 cells/well. After 28 h [ 34 ], cells were fixed in 70% ethanol [ 48 ], and DNA was stained using PI in the presence of DNase-free RNase A (40 mg/mL) for 30 min at 37 °C in the dark circumstance. Cell cycle analysis was performed using flow cytometry (Beckman Coulter FC500; Beckman Coulter) according to the manufacturer’s instructions.…”

“…Then 100 ng psi-check2 vector was transfected into the CHO cells in triplicate. 24, 72 and 120 h after transfection [ 34 ], cells were lysed utilizing passive lysis buffer containing Tris/phosphate 25 mM, Triton X100 1%, Glycerol 10%, and DTT 2 mM. After that the firefly intensity was measured using GloMax Multi-detection system (Promega) in a luminometer at the excitation wave length of 520 nm.…”

Affecting HEK293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes

“…The knockdown of INTS1, HNRHPC, OAZ, CASP8AP2 and PPP2R1A consistently improved the expression of at least two reporter proteins tested by up to 72% [ 115 ]. In follow-up studies, the CRISPR/Cas9 knockout of the ornithine decarboxylase antizyme1 ( OAZ1 ) resulted in a 5-fold improvement in luciferase production and a 2.5-fold improvement in transient specific secreted alkaline phosphatase (SEAP) production [ 116 ], and knockout of the caspase 8 associated protein 2 ( CASP8AP2 ) gene resulted in a 7-fold increase in specific luciferase production and a 2.4-fold increase in transient specific SEAP production [ 117 ].…”