nu-7026 and Hepatitis-B--Chronic

Chronic hepatitis B is a severe liver disease caused by hepatitis B virus (HBV) infection. Covalently closed circular DNA (cccDNA), a super-spiralized, double-stranded form of the HBV genome, is the major determinant of viral persistence. CRISPR/Cas9 nucleases have been recently shown to introduce double-stranded DNA breaks into HBV cccDNA. The inflicted damage results predominantly in erroneous repair of cccDNA by non-homologous end-joining (NHEJ). NHEJ has been suggested to enhance anti-HBV activity of CRISPR/Cas9 and increase cccDNA mutation. In this study, we assessed anti-HBV activity of CRISPR/Cas9 and cccDNA repair outcomes in an altered NHEJ/HR environment. NU7026, a strong inhibitor of NHEJ, prevented CRISPR/Cas9-mediated degradation of cccDNA and resulted in frequent on-target deletions. We conclude that CRISPR/Cas9 is a highly effective tool to degrade cccDNA and first demonstrate that inhibiting NHEJ impairs cccDNA degradation.

Topics: Apoptosis; Catalytic Domain; Cell Survival; Chromones; CRISPR-Cas Systems; DNA End-Joining Repair; DNA Mutational Analysis; DNA, Circular; DNA, Viral; Gene Deletion; Genome, Viral; Hep G2 Cells; Hepatitis B virus; Hepatitis B, Chronic; High-Throughput Nucleotide Sequencing; Humans; Morpholines; RNA, Guide, Kinetoplastida