CRISPR-Cas9 Mediated Knockout of Host Restriction Factors Allows Increasing Influenza A Virus Replication in Human Cell Line

The aim of presented work was exploring the CRISPR-Cas9 mediated knockout of several genes as a way to overcome the host restriction barrier and generate human cell line permissive to influenza infection. We selected some factors including AnxA6 and IRF7 which were earlier described as antivirus system components in human cells. By CRISPR-Cas9 genome editing we obtained several clones of 293FT cells with knockout of these genes. The suppression of target gene expression levels was confirmed with RT-PCR, western-blotting and RNA-Seq analysis. We compared replication dynamics of several influenza viruses in original and mutated cells in growth curve experiments. Using TCID50 assay, flow cytometry and immunofluorescence staining we showed that accumulation of influenza A virus in the mutant 293FT-ANXA6-/- and 293FT-IRF7-/- cells significantly exceeded the virus titer in the original 293FT cells. Transcriptome analysis was performed to reveal molecular pathways activated in intact and mutant cells during influenza infection. In addition to the upregulation of antiviral response pathways, RNASeq showed the activation of cholesterol metabolism, cell cycle regulation and chromatin remodeling pathways, as well as the downregulation of RNA modification processes. The individual knockout of host cell factors allows describing their role in virus replication control. We propose that multiple gene knockout could provide the desired level of influenza virus replication in adherent 293FT to use this cell line for influenza virus isolation and characterization.

This work was supported by Russian Science Foundation grant 18-75-10069.