International Session(Workshop)1(JSGE・JSH・JSGCS)
Thu. November 2nd   14:30 - 17:00   Room 11: Portopia Hotel South Wing Topaz
Identification of host factors associated with HBV infection by genome-wide CRISPR/Cas9 screen
Tadashi Inuzuka1,2, Atsushi Takai1, Hiroshi Seno1
1Department of Gastroenterology and Hepatology, Kyoto University Hospital, 2National Institute of Diabetes and Digestive and Kidney Diseases Liver Disease Branch
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.
Index Term 1: HBV
Index Term 2: gene screening
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