• Efficient and Affordable Genome Editing Protocol for Concurrent Caspase 8 Associated Protein 2 Gene Knock-in/Out in Chinese Hamster Ovary Cells using CRISPR-Cas9 System and All-in-One Technology
  • 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: Hosting more than one-third of biopharmaceuticals makes Chinese Hamster Ovary (CHO) cells an attractive target for genome editing technologies to produce cell lines with high yields of recombinant protein production. Modifying genes involved in apoptosis, specifically those related to initiation of apoptosis such as Caspases 8 Associated Protein 2 (CASP8AP2), may have a positive impact on the productivity of CHO cells. Innovation and the joining of various robust strategies to the CRISPR-Cas9 system pave the way for using this technology in CHO cell engineering. Objectives: The aim of this study is to introduce an efficient and time/cost-effective protocol using homology-independent targeted integration (HITI) strategy to perform simultaneous knock in/out in a CASP8AP2 gene of the CHO cell and assess the effect of this silencing on the CHO cell productivity.
  • Methods: We introduce an efficient protocol for CHO cell engineering using CRISPR/Cas9 system joined HITI strategy. Using a manual selection system eases and speeds up single-cell cloning. Limitation in designing efficient gRNAs in gene coding frame, we targeted a 3’ UTR of CASP8AP2 gene. In the following, the effect of this gene silencing on the expression of JRed protein in comparison with native CHO cells was investigated using flowcytometry.
  • Results: findings of this study displayed that CRISPR-Cas9 system joined HITI strategy provides a robust editing approach. The efficacy of this method was confirmed by achieving high efficiency of 60% knock-out clones. However, some of these clones were heterozygote, but most of them were homozygote. No need for any complex instruments except a fluorescent inverted microscope makes this protocol possible in a laboratory with low financial and instrumental resources. In addition, the flowcytometric analysis of protein expression revealed a 2.3-fold increase in JRed expression in CASP8AP2 silenced CHO cells compared to native cells.
  • Conclusion: We establish a straightforward procedure for gene modification in CHO cells which resulted in the generation of knockout CHO cells with higher productivity compared to native ones. Targeted integration of the interested gene expression cassettes in a site-specific manner as well as the possibility of labelling the knock-out cells for animal studies are the proses of this strategy.
  • Keywords: Chinese Hamster Ovary cells, CRISPR-Associated Protein 9, CASP8AP2,3’ UTR