Cellular and Molecular Life Sciences

, Volume 76, Issue 9, pp 1779–1794 | Cite as

Orthologous CRISPR/Cas9 systems for specific and efficient degradation of covalently closed circular DNA of hepatitis B virus

  • Dmitry KostyushevEmail author
  • Sergey Brezgin
  • Anastasiya Kostyusheva
  • Dmitry Zarifyan
  • Irina Goptar
  • Vladimir Chulanov
Original Article


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.


Antiviral Therapeutics NHEJ Mutations Cure Liver 



We thank Konstantin Severinov and Dieter Glebe for their helpful contributions, Yurii Babin and Konstantin Flyagin for technical assistance, and Vladimir Simirskii for access to microscopy.

Author contributions

DK, SB, and AK conducted all experiments; DZ and SB generated recombinant cccDNA and created gRNAs; DZ analyzed off-target sites and designed specific primers; IG conducted sequencing; DK conceived the project; AK helped conceive experiments with mutant gRNAs; DK, DZ, SB, and AK processed the data; DK wrote the manuscript; VC guided the study and revised the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no potential conflicts of interests. The authors have applied for patents concerning the use of Cas9 proteins and gRNAs for HBV therapy.


This work was supported by RSF Grant no. 16-15-10426.

Supplementary material

18_2019_3021_MOESM1_ESM.docx (2.5 mb)
Supplementary material 1 (DOCX 2524 kb)


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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Viral Hepatitis LaboratoryCentral Research Institute of EpidemiologyMoscowRussian Federation
  2. 2.Institute of ImmunologyFederal Medical Biological AgencyMoscowRussian Federation
  3. 3.Izmerov Research Institute of Occupational HealthMoscowRussian Federation
  4. 4.Sechenov UniversityMoscowRussian Federation

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