Sci Rep. 2019 Feb 12;9(1):1847. doi: 10.1038/s41598-019-38526-6.
Suppressing the NHEJ pathway by DNA-PKcs inhibitor NU7026 prevents degradation of HBV cccDNA cleaved by CRISPR/Cas9.
Kostyushev D1, Kostyusheva A2, Brezgin S2,3, Zarifyan D2, Utkina A2, Goptar I2,4, Chulanov V2,5.
Author information
1
Central Research Institute of Epidemiology, Viral Hepatitis, Moscow, 111123, Russian Federation. [email protected].
2
Central Research Institute of Epidemiology, Viral Hepatitis, Moscow, 111123, Russian Federation.
3
Institute of Immunology, Federal Medical Biological Agency, Moscow, 115478, Russian Federation.
4
Izmerov Research Institute of Occupational Health, Gene Engineering and Biotechnology, Moscow, 105275, Russian Federation.
5
I.M. Sechenov First Moscow State Medical University, Infectious Diseases, Moscow, 119146, Russian Federation.
Abstract
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.