Targeted HBx gene editing by CRISPR/Cas9 system effectively reduces epithelial to mesenchymal transition and HBV replication in hepatoma cells

objective: Hepatitis B virus X protein (HBx) play a key role in pathogenesis of HBV-induced hepatocellular carcinoma (HCC) by promoting epithelial to mesenchymal transition (EMT). In this study, we hypothesized that inhibition of HBx is an effective strategy to combat HCC. results: We designed and synthesized novel HBx gene specific single guide RNA (sgRNA) with CRISPR/Cas9 system and studied its in vitro effects on tumour properties of HepG2-2.15. Full length HBx gene was excised using HBx-CRISPR that resulted in significant knockdown of HBx expression in hepatoma cells. HBx-CRISPR also decreased levels of HBsAg and HBV cccDNA expression. A decreased expression of mesenchymal markers, proliferation and tumorigenic properties was observed in HBx-CRISPR treated cells as compared to controls in both two- and three- dimensional (2D and 3D) tumour models. Transcriptomics data showed that out of 1159 differentially expressed genes in HBx-CRISPR transfected cells as compared to controls, 70 genes were upregulated while 1089 genes associated with cell proliferation and EMT pathways were downregulated. conclusions: Thus, targeting of HBx by CRISPR/Cas9 gene editing system reduces covalently closed circular DNA (cccDNA) levels, HBsAg production and mesenchymal characteristics of HBV-HCC cells. We envision inhibition of HBx by CRISPR as a novel therapeutic approach for HBV-induced HCC.