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Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells
Published on: January 5, 2018
Targeting Caspase-3 Gene in rCHO Cell Line by CRISPR/Cas9 Editing Tool and Its Effect on Protein Production in
Amirabbas Rahimi1,2, Morteza Karimipoor2, Reza Mahdian2
1Laboratory of Regenerative Medicine and Biomedical Innovations, Department of National Cell Bank, Pasteur Institute of Iran, Tehran, Iran.
Background:
Chinese hamster ovary (CHO) cells are the widely used mammalian cell host for biopharmaceutical manufacturing. During cell cultures, CHO cells lose viability mainly from apoptosis. Inhibiting cell death is useful because prolonging cell lifespans can direct to more productive cell culture systems for biotechnology requests.
Objectives:
This study exploited a CRISPR/Cas9 technology to generate site-specific gene disruptions in the caspase-3 gene in the apoptosis pathway, which acts as an apoptotic regulator to extend cell viability in the CHO cell line.
Methods:
The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 system. The guide RNAs targeting the caspase-3 gene were designed, and vectors containing sgRNA and Cas9 were transfected into CHO cells that expressed erythropoietin as a heterologous protein. Indel formation was investigated by DNA sequencing. Caspase-3 expression was quantified by real-time PCR and western blot. The effect of editing the caspase-3 gene on the inhibition of apoptosis was also investigated by induction of apoptosis in manipulated cell lines by oleuropein. Finally, the erythropoietin production in the edited cells was compared to the control cells.
Results:
The caspase-3 manipulation significantly prolongation of the cell viability and decreased the caspase-3 expression level of protein in manipulated CHO cells (more than 6-fold, P-value < 0.0001). Manipulated cells displayed higher threshold tolerance to apoptosis compared to the control cells when they were induced by oleuropein. They show a higher IC50 than the control ones (7271 µM/mL Vs. 5741 µM/mL). They also show a higher proliferation rate than the control cells in the presence of an apoptosis inducer (P-value < 0.0001). Furthermore, manipulated cell lines significantly produce more recombinant protein in the presence of 2,000 µM oleuropein compared to the control ones (P-value = 0.0021).
Conclusions:
We understood that CRISPR/Cas9 could be effectively applied to suppress the expression of the caspase-3 gene and rescue CHO cells from apoptosis induced by cell stress and metabolites. The CRISPR/Cas9 system-assisted caspase-3 gene ablation can potentially increase erythropoietin yield in CHO cells.
Insights
CRISPR/Cas9 gene editing successfully suppressed caspase-3 expression in Chinese hamster ovary (CHO) cells, significantly extending cell viability and enhancing recombinant protein production. This approach offers a promising strategy for improving biopharmaceutical manufacturing processes.
Area of Science:
- Biotechnology
- Molecular Biology
- Cell Biology
Background:
- Chinese hamster ovary (CHO) cells are crucial for biopharmaceutical manufacturing.
- Apoptosis (programmed cell death) limits cell viability and productivity in CHO cell cultures.
- Extending cell lifespan is key to improving biotechnological yields.
Purpose of the Study:
- To utilize CRISPR/Cas9 gene editing to disrupt the caspase-3 gene in CHO cells.
- To enhance CHO cell viability and productivity by inhibiting apoptosis.
- To assess the impact of caspase-3 gene disruption on erythropoietin production.
Main Methods:
- Identified key pro-apoptotic genes using the STRING database.
- Designed and implemented CRISPR/Cas9 system targeting the caspase-3 gene in CHO cells producing erythropoietin.
- Quantified gene editing efficiency via DNA sequencing, real-time PCR, and Western blot.
- Assessed apoptosis inhibition and cell proliferation under induced stress.
- Compared erythropoietin production in edited vs. control cells.
Main Results:
- Caspase-3 expression was significantly reduced (over 6-fold) in edited CHO cells (P < 0.0001).
- Edited cells exhibited increased tolerance to apoptosis induction and higher proliferation rates.
- Enhanced IC50 values and improved recombinant erythropoietin yield were observed in manipulated cells.
- Cell viability was significantly prolonged in caspase-3 edited CHO cells (P < 0.0001).
Conclusions:
- CRISPR/Cas9 effectively suppresses caspase-3 expression, rescuing CHO cells from apoptosis.
- Caspase-3 gene ablation enhances cell viability and recombinant protein production in CHO cell lines.
- This gene editing strategy holds potential for increasing erythropoietin yield in biomanufacturing.
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