Amirabbas Rahimi, Morteza Karimipoor, Reza Mahdian, Atefeh Alipour, Saadi Hosseini, Marzieh Mohammadi, Hooman Kaghazian, Abdolrahim Abbasi, Hosein Shahsavarani, Mohammad Ali Shokrgozar
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The single guide RNAs (sgRNAs) targeting identified gene (BAX) were designed, and CHO cells were then transfected with vectors. Afterward, changes in the expression of the Bax gene and consequent production rates of erythropoietin were investigated in manipulated cells, even in the presence of an apoptosis inducer agent, oleuropein.</p><p><strong>Results: </strong>BAX disruption significantly prolonged cell viability and increased proliferation rate in manipulated clones (152%, P-value = 0.0002). This strategy reduced the levels of Bax protein expression in manipulated cells by more than 4.3-fold (P-value <0.0001). The Bax-8 manipulated cells displayed higher threshold tolerance to the stress and consequence apoptosis compared to the control group. Also, they exhibited a higher IC50 compared to the control in the presence of oleuropein (5095 µM.ml<sup>-1</sup> Vs. 2505 µM.ml<sup>-1</sup>). We found a significant increase in recombinant protein production levels in manipulated cells, even in the presence of 1,000 µM oleuropein compared to the control cell line (p-value=0.0002).</p><p><strong>Conclusions: </strong>CRISPR/Cas9 assisted BAX gene ablation is promising to improve erythropoietin production in CHO cells via engineering anti-apoptotic genes. 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引用次数: 0
摘要
背景:尽管最近在利用CHO细胞生产重组生物治疗药物方面取得了进展，但其生产力仍然低于工业需求，主要是由于细胞凋亡。目的:本研究旨在利用CRISPR/Cas9技术特异性破坏BAX基因，以减轻产生促红细胞生成素的重组中国仓鼠卵巢细胞的凋亡。材料和方法:利用STRING数据库鉴定CRISPR/Cas9技术修饰的促凋亡关键基因。设计靶向鉴定基因BAX的单导rna (single guide rna, sgRNAs)，转染CHO细胞。随后，在操纵细胞中研究了Bax基因表达的变化和随之而来的促红细胞生成素的产生率，甚至在存在凋亡诱导剂橄榄苦苷的情况下。结果:BAX破坏显著延长了操纵克隆的细胞活力和增殖率(152%，p值= 0.0002)。该策略使操纵细胞中的Bax蛋白表达水平降低了4.3倍以上(p值-1 Vs. 2505µM.ml-1)。我们发现，与对照细胞系相比，即使在存在1,000µM橄榄苦苷的情况下，操纵细胞的重组蛋白生产水平也显著增加(p值=0.0002)。结论:CRISPR/Cas9辅助BAX基因消融有望通过工程抗凋亡基因改善CHO细胞的促红细胞生成素生成。因此，有人建议利用CRISPR/Cas9等基因组编辑工具来开发宿主细胞，从而实现安全、可行、稳健的制造操作，并达到工业要求的产量。
Efficient CRISPR/Cas9-Mediated BAX Gene Ablation in CHO Cells To Impair Apoptosis and Enhance Recombinant Protein Production.
Background: Despite recent advances in recombinant biotherapeutics production using CHO cells, their productivity remains lower than industrial needs, mainly due to apoptosis.
Objectives: Present study aimed to exploit CRISPR/Cas9 technology to specifically disrupt the BAX gene to attenuate apoptosis in recombinant Chinese hamster's ovary cells producing erythropoietin.
Materials and methods: The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 technique. The single guide RNAs (sgRNAs) targeting identified gene (BAX) were designed, and CHO cells were then transfected with vectors. Afterward, changes in the expression of the Bax gene and consequent production rates of erythropoietin were investigated in manipulated cells, even in the presence of an apoptosis inducer agent, oleuropein.
Results: BAX disruption significantly prolonged cell viability and increased proliferation rate in manipulated clones (152%, P-value = 0.0002). This strategy reduced the levels of Bax protein expression in manipulated cells by more than 4.3-fold (P-value <0.0001). The Bax-8 manipulated cells displayed higher threshold tolerance to the stress and consequence apoptosis compared to the control group. Also, they exhibited a higher IC50 compared to the control in the presence of oleuropein (5095 µM.ml-1 Vs. 2505 µM.ml-1). We found a significant increase in recombinant protein production levels in manipulated cells, even in the presence of 1,000 µM oleuropein compared to the control cell line (p-value=0.0002).
Conclusions: CRISPR/Cas9 assisted BAX gene ablation is promising to improve erythropoietin production in CHO cells via engineering anti-apoptotic genes. Therefore, exploiting genome editing tools such as CRISPR/Cas9 has been proposed to develop host cells that result in a safe, feasible, and robust manufacturing operation with a yield that meets the industrial requirements.
期刊介绍：
Iranian Journal of Biotechnology (IJB) is published quarterly by the National Institute of Genetic Engineering and Biotechnology. IJB publishes original scientific research papers in the broad area of Biotechnology such as, Agriculture, Animal and Marine Sciences, Basic Sciences, Bioinformatics, Biosafety and Bioethics, Environment, Industry and Mining and Medical Sciences.
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