Department of Biomedical Science and Engineering, Konkuk University, Seoul 05029, Korea.
Int J Mol Sci. 2022 Jul 26;23(15):8217. doi: 10.3390/ijms23158217.
The current pandemic and the possible emergence of new viruses urgently require the rapid development of antiviral vaccines and therapeutics. However, some viruses or newly generated variants are difficult to culture in common cell types or exhibit low viral susceptibility in vivo, making it difficult to manufacture viral vector-based vaccines and understand host-virus interactions. To address these issues, we established new cell lines deficient in both type I and type II interferon responses, which are essential for host immunity and interference with virus replication. These cell lines were generated by developing an integrated CRISPR-Cas9 system that simultaneously expresses dual-guide RNA cassettes and Cas9 nuclease in a single plasmid. Using this highly efficient gene-editing system, we successfully established three cell lines starting from IFN-α/β-deficient Vero cells, deleting the single interferon-gamma () gene, the IFNG receptor 1 () gene, or both genes. All cell lines clearly showed a decrease in IFN-γ-responsive antiviral gene expression and cytokine production. Moreover, production of IFN-γ-induced cytokines remained low, even after HSV-1 or HCoV-OC43 infection, while expression of the receptor responsible for viral entry increased. Ultimately, knockout of IFN-signaling genes in these cell lines promoted cytopathic effects and increased apoptosis after viral infection up to three-fold. These results indicate that our integrated CRISPR-Cas9-mediated and -knockout cell lines promote virus replication and will be useful in viral studies used to design novel vaccines and therapies.
当前的大流行病和新病毒的可能出现迫切需要快速开发抗病毒疫苗和疗法。然而，一些病毒或新产生的变体难以在常见细胞类型中培养，或者在体内表现出低病毒易感性，使得制造基于病毒载体的疫苗和了解宿主-病毒相互作用变得困难。为了解决这些问题，我们建立了缺乏 I 型和 II 型干扰素反应的新型细胞系，这对于宿主免疫和干扰病毒复制至关重要。这些细胞系是通过开发一种集成的 CRISPR-Cas9 系统建立的，该系统在单个质粒中同时表达双向导 RNA 盒和 Cas9 核酸酶。使用这种高效的基因编辑系统，我们从 IFN-α/β 缺陷的 Vero 细胞开始成功建立了三个细胞系，分别敲除了单个干扰素-γ（IFN-γ）基因、IFN-γ 受体 1（IFNGR1）基因或这两个基因。所有细胞系均明显显示出 IFN-γ 反应性抗病毒基因表达和细胞因子产生减少。此外，即使在 HSV-1 或 HCoV-OC43 感染后，IFN-γ 诱导的细胞因子的产生仍然较低，而负责病毒进入的受体的表达增加。最终，这些细胞系中 IFN 信号基因的敲除促进了病毒感染后的细胞病变效应和细胞凋亡增加了三倍。这些结果表明，我们的集成 CRISPR-Cas9 介导的 和 -敲除细胞系促进了病毒复制，将有助于设计新型疫苗和疗法的病毒研究。
