Chronic hepatitis B virus (HBV) infection is the leading cause of hepatocellular carcinoma (HCC) world-wide. The mechanisms of HBV-mediated hepatocyte transformation and tumorigenesis remain largely unknown, due to the absence of an in vitro model recapitulating these processes. In this project, Hans Clevers will build on his recent demonstration of long-term in vitro HBV infection of human hepatocyte organoids. Taking advantage of efficient CRISPR-based gene editing of hepatocyte organoids, these HBV+ hepatocyte organoids will be further modified by mutations of HBV-associated cancer genes to sequentially acquire HCC phenotypes. These genetically modified HBV-associated HCC organoids will bring insight into HBV-driven malignant transformation and provide a flexible and robust platform to perform drug screening. This project will create and validate a unique long-term in vitro HCC-HBV infection model. Ultimately, this will allow us to understand the process and mechanisms of tumor transformation and establish a screening platform for HBV-associated HCC drugs.