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.

Knockout of IRF3 and IRF7 genes by CRISPR/Cas9 technology enhances porcine virus replication in the swine testicular (ST) cell line

“…[ 16 ] For instance, when the interferon‐gamma (IFNG) gene and the IFNG receptor 1 (IFNGR1) gene were knocked out in Vero cells, the virus titers of HSV‐1 or HCoV‐OC43 were promoted about 10‐fold. [ 17 ] Knocking out IRF7 in MDCK cells promoted the virus titers of the PR8 virus by 2‐5‐fold. [ 18 ] The ST IRF3/7 KO cell line we created was able to increase virus titers by around 5‐20‐fold.…”

Genomic Mutations of the STAT5 Transcription Factor Are Associated with Human Cancer and Immune Diseases

“…The efficiency of intracellular delivery of the CRISPR-Cas system have been improved by developing a smaller version of Cas9 endonuclease, such as CjCas, to fit the genome capacity of an adeno-associated viral vector, or by utilizing a baculoviral vector with a packaging capacity over 100 kb [ 88 , 89 ]. To further improve the mutation rate, a single plasmid expressing gRNA cassettes and Cas9 nuclease has been developed as an alternative to introducing gRNA and Cas9 into cells separately [ 90 ]. These strategies could facilitate the development of clinical therapeutics to treat STAT5 genetic mutation-driven diseases.…”

Animal Cell Lines as Expression Platforms in Viral Vaccine Production: A Post Covid-19 Perspective

“…In another study conducted by Manohar et al (2008), it was determined that the tumorigenicity of Vero cultures with passage number more than 200 increased depending on the passage number . In addition to all this, with a large number of cell engineering studies, especially using the CRISPR/Cas9 system, viral susceptibility was increased by knocking out various genes in Vero cells, and a significant increase in viral titers was observed in experiments with various viruses using edited cells. , …”

“…132 In addition to all this, with a large number of cell engineering studies, especially using the CRISPR/Cas9 system, viral susceptibility was increased by knocking out various genes in Vero cells, and a significant increase in viral titers was observed in experiments with various viruses using edited cells. 133,134 5.5. MDCK.…”