• Enhancing Apoptosis Resistance in Chinese Hamster Ovary (CHO) Cells through CRISPR/Cas9-Mediated Caspase 8 Associated Protein 2 Gene Editing
  • Soofia Sorourian,1 Abbas Behzad Behbahani,2,* Mohsen Forouzanfar,3 Mojtaba Jafarinia,4
    1. Department of Biology, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran
    2. Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
    3. Department of Biology, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran
    4. Department of Biology, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran


  • Introduction: Chinese Hamster Ovary (CHO) cells are of great interest for the production of biological drugs on a commercial scale. They are able to produce high levels of recombinant proteins, which are an important part of protein therapy and protein engineering. New genome editing technologies, such as CRISPR/Cas9, offer the potential to enhance CHO cell lines by modifying genes involved in apoptosis, the process of programmed cell death. This is because apoptosis is a major factor that limits the production of recombinant proteins in CHO cells. Objective This study aimed to investigate the role of the Caspase 8 Associated Protein 2 gene (CASP8AP2) gene in the apoptosis pathway. The CASP8AP2 gene is involved in the induction of apoptosis, and its knockout by the CRISPR/Cas9 system could lead to cells with increased resistance to apoptosis. This would allow for the production of higher levels of recombinant proteins in CHO cells.
  • Methods: We have developed a protocol for engineering CHO cells using the CRISPR/Cas9 system in conjunction with the homology-independent targeted integration (HITI) strategy, in which CHO cells constantly express GFP, thereby facilitating clone selection by deleting the CASP8AP2 gene. Apoptosis was assessed using flow cytometry to evaluate the effect of CASP8AP2 gene silencing on the viability of engineered cells, and the number of cells that underwent early apoptosis, late apoptosis, and necrosis compared to parental CHO cells.
  • Results: Our study shows that the CASP8AP2 gene, which plays a role in stimulating extrinsic apoptosis, its deletion by the CRISPR-Cas system causes resistance to apoptosis in cells lacking CASP8AP2, which shows that this approach has a significant potential to improve the performance of CHO cells. While some clones showed heterozygosity, more than 60% of the knockout clones were successfully homozygous. This protocol does not require very complicated equipment only relies on an inverted fluorescent microscope, and allows implementation in laboratories with limited financial resources and instruments. Flow cytometry analysis showed that the deletion of the CASP8AP2 gene causes cells to become resistant to apoptosis. It can be of great help in the production of therapeutic proteins and the expression of recombinant proteins in CHO cells in the future.
  • Conclusion: The developed protocol provides an efficient and cost-effective tool for simultaneous knock-in/knockout of CASP8AP2 gene in CHO cells. Successful gene editing in apoptosis resistance subsequently increases protein expression, underscoring the potential for better biopharmaceutical production. These findings contribute to the advancement of CHO cell engineering and its applications in optimizing the yield of recombinant protein.
  • Keywords: CHO cells, CRISPR-Associated Protein 9, Homology-independent targeted integration , GFP