A CRISPR-HITI strategy approach to improve CHO cell viability by modifying the 3'UTR of Caspase 8 Associated Protein 2.

Abstract

Chinese Hamster Ovary (CHO) cells are essential in biopharmaceutical manufacturing. Scientists use CRISPR to enhance productivity. mRNAs contain UTRs that regulate gene expression, affecting protein abundance. Targeting these regions creates desirable knockout cells. The Caspase 8 Associated Protein 2 (CASP8AP2) gene is a promising target for improving host cell viability. This study used the CRISPR-Homology-Independent Targeted Integration (HITI) strategy to modify the 3'UTR region of the CASP8AP2 gene in CHO cells. The aim was to evaluate the effects of CASP8AP2 silencing on cell proliferation, viability, apoptosis, and the cell cycle. CASP8AP2 silencing was assessed post-modification by extracting genomic DNA from modified and unmodified CHO cells, followed by PCR and sequencing to confirm deletions. Cell proliferation and viability were measured using MTT assays, and cell cycle analysis was performed via flow cytometry. Apoptosis was evaluated through Annexin V/PE staining and flow cytometry, with apoptosis resistance assessed by determining the IC₅₀ of sodium butyrate. Results showed CASP8AP2 deletion did not affect cell proliferation or the cell cycle but improved CHO cell viability and increased resistance to apoptosis. The IC₅₀ for sodium butyrate was higher in CASP8AP2 knockout cells (7.84 mM) compared to native cells (3.43 mM), indicating enhanced apoptosis resistance. This study highlights CASP8AP2's role in apoptosis regulation without impacting cell proliferation or the cell cycle. CASP8AP2 deletion enhances viability and resistance to apoptosis, suggesting it as a target for improving recombinant protein production. Further research is needed to elucidate the molecular mechanisms and develop therapeutic strategies based on this approach.