Hepatitis B virus is a DNA virus that infects the liver. Current drugs efficiently suppress viral replication but do not clear the viral episomal DNA (cccDNA), and therefore rarely cure patients. The discovery of CRISPR (clustered regularly interspersed short palindromic repeat) as a bacterial adaptive immune system, and subsequent engineering of this system to precisely cleave DNA, provides a potential approach for direct targeting of HBV DNA in infected cells. We engineered a set of CRISPR/Cas9 guide RNAs targeting conserved regions in the HBV genome and screened for their utility in-vitro and in-vivo. We identified three guide RNAs highly potent for HBV inhibition that were further tested in hepatoma cells, which stably express HBV from an integrated transgene and also maintain a pool of cccDNA. A kinetic analysis revealed a robust reduction in HBV replicative forms as well as in cccDNA levels over time. Analysis of CRISPR-mediated DNA cleavage revealed substantial cleavage presence in integrated HBV DNA but much less in residual cccDNA, suggesting that cccDNA targeted by CRISPR/Cas9 may be rapidly degraded upon cleavage rather than repaired. Finally, hepatoma cells over expressing the HBV receptor NTCP and selected for the expression of HBV specific CRISPR/Cas9 guide RNAs were infected with HBV. A large attenuation of HBV infection was observed in CRISPR/Cas9 expressing cells, confirming the utility of this system in the context of natural infection. Thus, the CRISPR/Cas9 system suppresses HBV and possibly eliminates cccDNA. This study highlights the possible utility of this approach as a curative anti-HBV therapy.
