In line with our interest in HBV biology, the goal of our current research effort is to investigate one of the least understood steps in HBV replication: the mechanism by which the viral genome is converted into a covalently closed circular (ccc) DNA form and how intracellular amplification of cccDNA is regulated. To conduct our studies, we have developed a CRISPR/Cas9 platform permitting HBV infection of cells with specific gene knockouts. In addition, we are exploring a novel strategy to eliminate cccDNA from infected cells with the help of the CRISPR/Cas9 system. Finally, we are developing methods to visualize individual copies of cccDNA in HBV infected cells. We seek to determine the localization of cccDNA in nuclei of infected cells and to monitor the fate of cccDNA under conditions mimicking natural recovery from acute HBV infections. Our long-term goal is to provide novel insights into the biology of cccDNA that can be used for the development of therapies to cure chronic hepatitis B.