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Molecular Biology

, Volume 53, Issue 2, pp 274–285 | Cite as

Small Molecular Inhibitors of DNA Double Strand Break Repair Pathways Increase the ANTI-HBV Activity of CRISPR/Cas9

  • A. P. Kostyusheva
  • D. S. KostyushevEmail author
  • S. A. Brezgin
  • D. N. Zarifyan
  • E. V. Volchkova
  • V. P. Chulanov
MOLECULAR CELL BIOLOGY

Abstract

The CRISPR/Cas9 nuclease system can effectively suppress the replication of the hepatitis B virus (HBV), while covalently closed circular DNA (cccDNA), a highly resistant form of the virus, persists in the nuclei of infected cells. The most common outcome of DNA double-strand breaks (DSBs) in cccDNA caused by CRISPR/Cas9 is double-strand break repair by nonhomologous end-joining, which results in insertion/deletion mutations. Modulation of the DNA double-strand break repair pathways by small molecules was shown to stimulate CRISPR/Cas9 activity and may potentially be utilized to enhance the elimination of HBV cccDNA. In this work, we used inhibitors of homologous (RI-1) and nonhomologous (NU7026) end-joining and their combination to stimulate antiviral activity of CRISPR/Cas9 on two cell models of HBV in vitro, i.e., the HepG2-1.1merHBV cells containing the HBV genome under the tet-on regulated cytomegalovirus promoter and the HepG2-1.5merHBV cells containing constitutive expression of HBV RNA under the wild-type promoter. The treatment of the cells with RI-1 or NU7026 after lentiviral transduction of CRISPR/Cas9 drops the levels of cccDNA compared to the DMSO-treated control. RI-1 and NU7026 resulted in 5.0–6.5 times more significant reduction in the HBV cccDNA level compared to the mock-control. In conclusion, the inhibition of both homologous and nonhomologous DNA double-strand break repair pathways increases the elimination of HBV cccDNA by CRISPR/Cas9 system in vitro, which may potentially be utilized as a therapeutic approach to treat chronic hepatitis B.

Keywords:

CRISPR/Cas9 hepatitis B virus covalently closed circular DNA lentiviral transduction HR NHEJ DNA double-strand breaks 

Notes

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Copyright information

© Pleiades Publishing, Inc. 2019

Authors and Affiliations

  • A. P. Kostyusheva
    • 1
  • D. S. Kostyushev
    • 1
    Email author
  • S. A. Brezgin
    • 1
    • 2
  • D. N. Zarifyan
    • 1
  • E. V. Volchkova
    • 3
  • V. P. Chulanov
    • 1
    • 3
  1. 1.Central Research Institute of Epidemiology, Federal Service for the Oversight of Consumer Protection and WelfareMoscowRussia
  2. 2.Institute of Immunology of Federal Medical-Biological AgencyMoscowRussia
  3. 3.Sechenov Moscow State Medical UniversityMoscowRussia

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