Time and Cost-Effective Genome Editing Protocol for Simultaneous Caspase 8 Associated Protein 2 Gene Knock in/out in Chinese Hamster Ovary Cells Using CRISPR-Cas9 System

Document Type : Research Paper


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


Background: CHO cells are preferred for producing biopharmaceuticals, and genome editing technologies offer
opportunities to enhance recombinant protein production. Targeting apoptosis-related genes, such as Caspases 8-Associated Protein 2 (CASP8AP2), improves CHO cell viability and productivity. Integrating robust strategies with the CRISPR-Cas9 system enables its application in CHO cell engineering.

Objectives: This study was performed to develop a cost-effective protocol using the CRISPR-Cas9 system combined
with the HITI strategy for simultaneous CASP8AP2 gene deletion/insertion in CHO cells and to assess its impact on cell
viability and protein expression.

Materials and Methods: We developed an efficient protocol for CHO cell engineering by combining CRISPR/Cas9
with the HITI strategy. Two distinct sgRNA sequences were designed to target the 3’ UTR region of the CASP8AP2 gene
using CHOPCHOP software. The gRNAs were cloned into PX459 and PX460-1 vectors and transfected into CHO cells
using the cost-effective PEI reagent. A manual selection system was employed to streamline the process of single-cell
cloning. MTT assays assessed gene silencing and cell viability at 24, 48, and 72 hours. Flow cytometry evaluated protein
expression in CASP8AP2-silenced CHO cells.

Results: The study confirmed the robustness of combining CRISPR-Cas9 with the HITI strategy, achieving a high
60% efficiency in generating knockout clones. PEI transfection successfully delivered the constructs to nearly 65%
of the clones, with the majority being homozygous. The protocol proved feasible for resource-limited labs, requiring
only an inverted fluorescent microscope. CASP8AP2 knockout (CHO-KO) cells exhibited significantly extended cell
viability compared to CHO-K1 cells when treated with NaBu, with IC50 values of 7.28 mM and 14.25 mM at 48 hours,
respectively (P-value: 24 hours = < 0.0001, 48 hours = < 0.0001, P-value: 72 hours = 0.0007). CHO CASP8AP2-silenced
cells showed a 1.3-fold increase in JRed expression compared to native cells.

Conclusions: CRISPR-Cas9 and HITI strategy was used to efficiently engineer CHO cells for simultaneous CASP8AP2
gene deletion/insertion, which improved cell viability and protein expression.


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