Inactivation of the key ORFs of HBV for antiviral therapy by non-cleavage base editing.

OBJECTIVESHepatitis B virus (HBV) infection is the key cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Currently available anti-HBV drugs are more or less defective owing to the unremovable covalently closed circular DNA (cccDNA). Thus, CRISPR/Cas9 is a promising therapeutic strategy for anti-HBV therapy. Double-strand breaks (DSBs) and uncontrolled genomic rearrangements occur inevitably. In this study, we aimed to use base editors to control HBV infection.

METHODSBase editors precisely instal targeted point mutations without requiring DSBs or donor DNA templates, and without relying on homology-directed repair (HDR) or nonhomologous end joining (NHEJ). Adenine base editors (ABEs) and cytosine base editors (CBEs) catalyse A• T to G •C and C• G to T •A conversions, respectively. In this study, to control HBV replication by modifying and inactivating key HBV genes, recently developed CRISPR/Cas-mediated SpRY-ABE8e and CBE4-max were utilised to falsify and invalidate the ATG initiation codons of the S, Pre-S1, PreS2, C, Pre-C, X, and P genes.

RESULTSThe ATG initiation codons of HBV genes were edited by ABE/CBE. The expected point mutations were successfully introduced, resulting in the simultaneous suppression of HBV antigen expression and replication to varying degrees.

CONCLUSIONSOur study focused on clearing HBV using base and provided experimental and theoretical evidence for the treatment of chronic HBV infection. Thus, base editing is a potential strategy for curing CHB by permanently inactivating the integrated DNA and cccDNA without using DSBs.