Integrated CRISPR-Cas9 System-Mediated Knockout of IFN-γ and IFN-γ Receptor 1 in the Vero Cell Line Promotes Viral Susceptibility

Abstract

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 (IFNG) gene, the IFNG receptor 1 (IFNGR1) 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 IFNG- and IFNGR1-knockout cell lines promote virus replication and will be useful in viral studies used to design novel vaccines and therapies.