| Innovative therapeutic strategies like host-targeting drugs have emerged as novel candidates for the treatment of HBV infection. To identify unknown potential host-dependency factors of HBV, we generated an HBV reporter virus, HBV-RFP, expressing red fluorescent protein after infection. We transduced HepG2NTCP/Cas9 cells with commercial pooled lentiviruses encoding guide RNA library targeting 19114 human genes. The edited cells were infected with HBV-RFP and 5% RFP-negative cells were then sorted by FACS. A separate set of HepG2NTCP/Cas9 was infected, not sorted and expanded as the control. To determine the knocked-out human genes that were enriched in comparison with the control, genomic DNA was extracted from these cells, PCR-amplified for the gRNAs, and subjected to next-generation sequencing. By MAGeCK analysis, we determined 63 genes with RPKM of 1 or higher including previously known HBV proviral factors such as RXRA, POLL, LDLR, and NTCP. To investigate the relationship between the 63 genes and HBV infection, we generated gene-KO cells for each gene by CRISPR/Cas9. These gene-KO cells were infected with HBV and analyzed for intracellular HBV RNA levels at 7 dpi. We selected 14 genes whose knock-out significantly decreased HBV infection in cells originated from HepG2NTCP/Cas9 cells. Further studies are being conducted to elucidate the mechanisms of interactions among these factors and HBV infection. |
