1887

Abstract

Hepatitis B virus (HBV) remains a global health threat as chronic HBV infection may lead to liver cirrhosis or cancer. Current antiviral therapies with nucleoside analogues can inhibit the replication of HBV, but do not disrupt the already existing HBV covalently closed circular DNA. The newly developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a powerful tool to target cellular genome DNA for gene editing. In order to investigate the possibility of using the CRISPR/Cas9 system to disrupt the HBV DNA templates, we designed eight guide RNAs (gRNAs) that targeted the conserved regions of different HBV genotypes, which could significantly inhibit HBV replication both and . Moreover, the HBV-specific gRNA/Cas9 system could inhibit the replication of HBV of different genotypes in cells, and the viral DNA was significantly reduced by a single gRNA/Cas9 system and cleared by a combination of different gRNA/Cas9 systems.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.000159
2015-08-01
2024-04-24
Loading full text...

Full text loading...

/deliver/fulltext/jgv/96/8/2252.html?itemId=/content/journal/jgv/10.1099/vir.0.000159&mimeType=html&fmt=ahah

References

  1. Ayoub W.S., Keeffe E.B. 2011; Review article: current antiviral therapy of chronic hepatitis B. Aliment Pharmacol Ther 34:1145–1158 [View Article][PubMed]
    [Google Scholar]
  2. Bloom K., Ely A., Mussolino C., Cathomen T., Arbuthnot P. 2013; Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases. Mol Ther 21:1889–1897 [View Article][PubMed]
    [Google Scholar]
  3. Cao L., Wu C., Shi H., Gong Z., Zhang E., Wang H., Zhao K., Liu S., Li S., other authors. 2014; Coexistence of hepatitis B virus quasispecies enhances viral replication and the ability to induce host antibody and cellular immune responses. J Virol 88:8656–8666 [View Article][PubMed]
    [Google Scholar]
  4. Chan H.L. 2011; Significance of hepatitis B virus genotypes and mutations in the development of hepatocellular carcinoma in Asia. J Gastroenterol Hepatol 26:8–12 [View Article][PubMed]
    [Google Scholar]
  5. Chudy M., Hanschmann K.M., Kress J., Nick S., Campos R., Wend U., Gerlich W., Nübling C.M. 2012; First WHO International Reference Panel containing hepatitis B virus genotypes A–G for assays of the viral DNA. J Clin Virol 55:303–309 [View Article][PubMed]
    [Google Scholar]
  6. Cradick T.J., Fine E.J., Antico C.J., Bao G. 2013; CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res 41:9584–9592 [View Article][PubMed]
    [Google Scholar]
  7. Ebina H., Misawa N., Kanemura Y., Koyanagi Y. 2013; Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep 3:2510 [View Article][PubMed]
    [Google Scholar]
  8. Gaj T., Gersbach C.A., Barbas C.F. III 2013; ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405 [View Article][PubMed]
    [Google Scholar]
  9. Hao R., He J., Liu X., Gao G., Liu D., Cui L., Yu G., Yu W., Chen Y., other authors. 2015; Inhibition of hepatitis B virus gene expression and replication by hepatocyte nuclear factor 6. J Virol 89:4345–4355 [CrossRef]
    [Google Scholar]
  10. Hsu P.D., Scott D.A., Weinstein J.A., Ran F.A., Konermann S., Agarwala V., Li Y., Fine E.J., Wu X., other authors. 2013; DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832 [View Article][PubMed]
    [Google Scholar]
  11. Hsu P.D., Lander E.S., Zhang F. 2014; Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278 [View Article][PubMed]
    [Google Scholar]
  12. Huang L.R., Wu H.L., Chen P.J., Chen D.S. 2006; An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection. Proc Natl Acad Sci U S A 103:17862–17867 [View Article][PubMed]
    [Google Scholar]
  13. Jablonowski H. 2003; IFN-alpha: therapeutic options in HIV and HIV/HCV, HIV/HBV dual infection. Infection 31:131–133[PubMed]
    [Google Scholar]
  14. Januszkiewicz-Lewandowska D., Rucka A., Kowala-Piaskowska A., Bereszynska I., Mozer-Lisewska I., Zajac-Spychala O., Wysocki J., Nowak J. 2014; Mutations in Pol gene of hepatitis B virus in patients with chronic hepatitis B before and after therapy with nucleoside/nucleotide analogues. Acta Virol 58:185–189 [View Article][PubMed]
    [Google Scholar]
  15. Joung J.K., Sander J.D. 2013; TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 14:49–55 [View Article][PubMed]
    [Google Scholar]
  16. Kennedy E.M., Bassit L.C., Mueller H., Kornepati A.V., Bogerd H.P., Nie T., Chatterjee P., Javanbakht H., Schinazi R.F., Cullen B.R. 2014; Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease. Virology 476:196–205 [View Article][PubMed]
    [Google Scholar]
  17. Li F., Zhang D., Li Y., Jiang D., Luo S., Du N., Chen W., Deng L., Zeng C. 2015; Whole genome characterization of hepatitis B virus quasispecies with massively parallel pyrosequencing. Clin Microbiol 21:280–287 [CrossRef]
    [Google Scholar]
  18. Lin S.R., Yang H.C., Kuo Y.T., Liu C.J., Yang T.Y., Sung K.C., Lin Y.Y., Wang H.Y., Wang C.C., other authors. 2014; The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo . Mol Ther Nucleic Acids 3:e186 [View Article][PubMed]
    [Google Scholar]
  19. Liu D., Wu A., Cui L., Hao R., Wang Y., He J., Guo D. 2014a; Hepatitis B virus polymerase suppresses NF-κB signaling by inhibiting the activity of IKKs via interaction with Hsp90β. PLoS One 9:e91658 [View Article][PubMed]
    [Google Scholar]
  20. Liu J., Kosinska A., Lu M., Roggendorf M. 2014b; New therapeutic vaccination strategies for the treatment of chronic hepatitis B. Virol Sin 29:10–16 [View Article][PubMed]
    [Google Scholar]
  21. Mani M., Kandavelou K., Dy F.J., Durai S., Chandrasegaran S. 2005; Design, engineering, and characterization of zinc finger nucleases. Biochem Biophys Res Commun 335:447–457 [View Article][PubMed]
    [Google Scholar]
  22. Perz J.F., Armstrong G.L., Farrington L.A., Hutin Y.J., Bell B.P. 2006; The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J Hepatol 45:529–538 [View Article][PubMed]
    [Google Scholar]
  23. Schädler S., Hildt E. 2009; HBV life cycle: entry and morphogenesis. Viruses 1:185–209 [View Article][PubMed]
    [Google Scholar]
  24. Seeger C., Sohn J.A. 2014; Targeting hepatitis B virus with CRISPR/Cas9. Mol Ther Nucleic Acids 3:e216 [View Article][PubMed]
    [Google Scholar]
  25. Tian Y., Chen W.L., Ou J.H. 2011; Effects of interferon-α/β on HBV replication determined by viral load. PLoS Pathog 7:e1002159 [View Article][PubMed]
    [Google Scholar]
  26. Weber N.D., Stone D., Sedlak R.H., De Silva Feelixge H.S., Roychoudhury P., Schiffer J.T., Aubert M., Jerome K.R. 2014; AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication. PLoS One 9:e97579 [View Article][PubMed]
    [Google Scholar]
  27. Werle-Lapostolle B., Bowden S., Locarnini S., Wursthorn K., Petersen J., Lau G., Trepo C., Marcellin P., Goodman Z., Delaney W.E. IV 2004; Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 126:1750–1758 [View Article][PubMed]
    [Google Scholar]
  28. Wilson A.C., Patient R.K. 1991; Evaluation of extrachromosomal gene copy number of transiently transfected cell lines. Methods Mol Biol 7:397–404[PubMed]
    [Google Scholar]
  29. Xie Y.H., Hong R., Liu W., Liu J., Zhai J.W. 2010; Development of novel therapeutics for chronic hepatitis B. Virol Sin 25:294–300 [View Article][PubMed]
    [Google Scholar]
  30. Yuen K.S., Chan C.P., Wong N.H., Ho C.H., Ho T.H., Lei T., Deng W., Tsao S.W., Chen H., other authors. 2015; CRISPR/Cas9-mediated genome editing of Epstein–Barr virus in human cells. J Gen Virol 96:626–636 [CrossRef]
    [Google Scholar]
  31. Zhen S., Hua L., Liu Y.H., Gao L.C., Fu J., Wan D.Y., Dong L.H., Song H.F., Gao X. 2015; Harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated Cas9 system to disrupt the hepatitis B virus. Gene Ther 22:404–412 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.000159
Loading
/content/journal/jgv/10.1099/vir.0.000159
Loading

Data & Media loading...

Supplements

Supplementary Data

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error