Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing

Area of Science:

• Molecular Biology
• Virology
• Gene Editing Technologies

Background:

• Chronic hepatitis B virus (HBV) infection persists due to resilient covalently closed circular DNA (cccDNA).
• CRISPR/Cas9 gene editing shows promise for HBV cure but risks host genome damage via double-strand breaks (DSBs).

Purpose of the Study:

• To evaluate CRISPR/Cas9-mediated base editors (BEs) for inactivating HBV gene expression without inducing DSBs.
• To assess the efficacy of BEs in targeting integrated HBV DNA and episomal cccDNA.

Main Methods:

• Screening of SpCas9-derived BEs and guide RNAs (gRNAs) for nonsense mutations in HBV genes.
• Testing BEs in cells with integrated HBV genomes and an in vitro HBV infection system.
• Assessing for insertions or deletions (indels) and gene expression changes post-editing.

Main Results:

• SpCas9-BE with specific gRNAs effectively edited HBV polymerase and surface genes, reducing viral gene expression in integrated HBV.
• Base editing induced minimal indels, avoiding significant host genome damage.
• Simultaneous suppression of polymerase and surface genes was observed with certain point mutations.
• Episomal cccDNA was successfully edited, suppressing viral gene expression in an in vitro HBV model.

Conclusions:

• CRISPR-mediated base editing presents a safe and effective strategy for chronic hepatitis B (CHB) treatment.
• This method offers a potential cure by permanently inactivating both integrated HBV DNA and cccDNA without host genome DSBs.