Cell Mol Life Sci. 2019 Jan 23. doi: 10.1007/s00018-019-03021-8. [Epub ahead of print]
Orthologous CRISPR/Cas9 systems for specific and efficient degradation of covalently closed circular DNA of hepatitis B virus.
Kostyushev D1, Brezgin S2,3, Kostyusheva A2, Zarifyan D2, Goptar I2,4, Chulanov V2,5.
Author information
1
Viral Hepatitis Laboratory, Central Research Institute of Epidemiology, 3A Novogireevskaya Street, Moscow, 111123, Russian Federation. [email protected].
2
Viral Hepatitis Laboratory, Central Research Institute of Epidemiology, 3A Novogireevskaya Street, Moscow, 111123, Russian Federation.
3
Institute of Immunology, Federal Medical Biological Agency, Moscow, 115478, Russian Federation.
4
Izmerov Research Institute of Occupational Health, Moscow, 105275, Russian Federation.
5
Sechenov University, Moscow, 119146, Russian Federation.
Abstract
Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence and chronic hepatitis B. CRISPR/Cas9 nucleases can specifically target HBV cccDNA for decay, but off-target effects of nucleases in the human genome limit their clinical utility. CRISPR/Cas9 systems from four different species were co-expressed in cell lines with guide RNAs targeting conserved regions of the HBV genome. CRISPR/Cas9 systems from Streptococcus pyogenes (Sp) and Streptococcus thermophilus (St) targeting conserved regions of the HBV genome blocked HBV replication and, most importantly, resulted in degradation of over 90% of HBV cccDNA by 6 days post-transfection. Degradation of HBV cccDNA was impaired by inhibition of non-homologous end-joining pathway and resulted in an erroneous repair of HBV cccDNA. HBV cccDNA methylation also affected antiviral activity of CRISPR/Cas9. Single-nucleotide HBV genetic variants did not impact anti-HBV activity of St CRISPR/Cas9, suggesting its utility in targeting many HBV variants. However, two or more mismatches impaired or blocked CRISPR/Cas9 activity, indicating that host DNA will not likely be targeted. Deep sequencing revealed that Sp CRISPR/Cas9 induced off-target mutagenesis, whereas St CRISPR/Cas9 had no effect on the host genome. St CRISPR/Cas9 system represents the safest system with high anti-HBV activity.
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