Skip to main content

Advertisement

Log in

CRISPR/Cas9-based tools for targeted genome editing and replication control of HBV

  • Review
  • Published:
Virologica Sinica

Abstract

Hepatitis B virus (HBV) infection remains a major global health problem because current therapies rarely eliminate HBV infections to achieve a complete cure. A different treatment paradigm to effectively clear HBV infection and eradicate latent viral reservoirs is urgently required. In recent years, the development of a new RNA-guided gene-editing tool, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system, has greatly facilitated site-specific mutagenesis and represents a very promising potential therapeutic tool for diseases, including for eradication of invasive pathogens such as HBV. Here, we review recent advances in the use of CRISPR/Cas9, which is designed to target HBV specific DNA sequences to inhibit HBV replication and to induce viral genome mutation, in cell lines or animal models. Advantages, limitations and possible solutions, and proposed directions for future research are discussed to highlight the opportunities and challenges of CRISPR/Cas9 as a new, potentially curative therapy for chronic hepatitis B infection.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Annoni A, Goudy K, Akbarpour M, Naldini L, Roncarolo MG. 2013. Immune responses in liver-directed lentiviral gene therapy. Transl Res, 161: 230–240.

    Article  CAS  PubMed  Google Scholar 

  • Arazoe T, Miyoshi K, Yamato T, Ogawa T, Ohsato S, Arie T, Kuwata S. 2015. Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus. Biotechnol Bioeng.

    Google Scholar 

  • Asokan A, Schaffer DV, Samulski RJ. 2012. The AAV vector toolkit: poised at the clinical crossroads. Mol Ther, 20: 699–708.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Beck J, Nassal M. 2007. Hepatitis B virus replication. World J Gastroenterol, 13: 48–64.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chen J, Yuan Z. 2014. Interplay between hepatitis B virus and the innate immune responses: implications for new therapeutic strategies. Virol Sin, 29: 17–24.

    Article  PubMed  Google Scholar 

  • Choi PS, Meyerson M. 2014. Targeted genomic rearrangements using CRISPR/Cas technology. Nat Commun, 5: 3728.

    PubMed Central  CAS  PubMed  Google Scholar 

  • Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science, 339: 819–823.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Datta S, Chatterjee S, Veer V, Chakravarty R. 2012. Molecular biology of the hepatitis B virus for clinicians. J Clin Exp Hepatol, 2: 353–365.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Dong C, Qu L, Wang H, Wei L, Dong Y, Xiong S. 2015. Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication. Antiviral Res, 118: 110–117.

    Article  CAS  PubMed  Google Scholar 

  • Doudna JA, Charpentier E. 2014. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 346: 1258096.

    Article  Google Scholar 

  • Dryden KA, Wieland SF, Whitten-Bauer C, Gerin JL, Chisari FV, Yeager M. 2006. Native hepatitis B virions and capsids visualized by electron cryomicroscopy. Mol Cell, 22: 843–850.

    Article  CAS  PubMed  Google Scholar 

  • Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM. 2013. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods, 10: 1116–1121.

    Article  CAS  PubMed  Google Scholar 

  • Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. 2013. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol, 31: 822–826.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. 2014. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol, 32: 279–284.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ganem D, Varmus HE. 1987. The molecular biology of the hepatitis B viruses. Annu Rev Biochem, 56: 651–693.

    Article  CAS  PubMed  Google Scholar 

  • Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadan AH, Moineau S. 2010. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 468: 67–71.

    Article  CAS  PubMed  Google Scholar 

  • Gebbing M, Bergmann T, Schulz E, Ehrhardt A. 2015. Gene therapeutic approaches to inhibit hepatitis B virus replication. World J Hepatol, 7: 150–164.

    Article  PubMed Central  PubMed  Google Scholar 

  • Hadziyannis SJ. 2014. Update on Hepatitis B Virus Infection: Focus on Treatment. J Clin Transl Hepatol, 2: 285–291.

    Article  PubMed Central  PubMed  Google Scholar 

  • Haft DH, Selengut J, Mongodin EF, Nelson KE. 2005. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol, 1: e60.

    Article  PubMed Central  PubMed  Google Scholar 

  • Hai H, Tamori A, Kawada N. 2014. Role of hepatitis B virus DNA integration in human hepatocarcinogenesis. World J Gastroenterol. 20: 6236–6243.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Horvath P, Barrangou R. 2010. CRISPR/Cas, the immune system of bacteria and archaea. Science, 327: 167–170.

    Article  CAS  PubMed  Google Scholar 

  • Isorce N, Lucifora J, Zoulim F, Durantel D. 2015. Immune-modulators to combat hepatitis B virus infection: From IFN-alpha to novel investigational immunotherapeutic strategies. Antiviral Res, 122: 69–81.

    Article  CAS  PubMed  Google Scholar 

  • Iwamoto M, Bjorklund T, Lundberg C, Kirik D, Wandless TJ. 2010. A general chemical method to regulate protein stability in the mammalian central nervous system. Chem Biol, 17: 981–988.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Karimova M, Beschorner N, Dammermann W, Chemnitz J, Indenbirken D, Bockmann JH, Grundhoff A, Luth S, Buchholz F, Schulze Zur, Wiesch J, Hauber J. 2015. CRISPR/Cas9 nickase- mediated disruption of hepatitis B virus open reading frame S and X. Sci Rep, 5: 13734.

    Article  PubMed Central  PubMed  Google Scholar 

  • Kennedy EM, Bassit LC, Mueller H, Kornepati AV, Bogerd HP, Nie T, Chatterjee P, Javanbakht H, Schinazi RF, Cullen BR. 2015a. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease. Virology, 476: 196–205.

    Article  CAS  PubMed  Google Scholar 

  • Kennedy EM, Cullen BR. 2015. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment. Virology, 479–480: 213–220.

    Article  PubMed  Google Scholar 

  • Kennedy EM, Kornepati AV, Mefferd AL, Marshall JB, Tsai K, Bogerd HP, Cullen BR. 2015b. Optimization of a multiplex CRISPR/Cas for use as an antiviral therapeutic. Methods. pii: S1046-2023(15)30052-9

    Google Scholar 

  • Komatsu H. 2014. Hepatitis B virus: where do we stand and what is the next step for eradication? World J Gastroenterol, 20: 8998–9016.

    Article  PubMed Central  PubMed  Google Scholar 

  • Koskella B. 2015. Research highlights for issue 6: the CRISPR/Cas revolution. Evol Appl, 8: 525–526.

    Article  PubMed Central  PubMed  Google Scholar 

  • Koumbi L. 2015. Current and future antiviral drug therapies of hepatitis B chronic infection. World J Hepatol, 7: 1030–1040.

    Article  PubMed Central  PubMed  Google Scholar 

  • Levrero M, Pollicino T, Petersen J, Belloni L, Raimondo G, Dandri M. 2009. Control of cccDNA function in hepatitis B virus infection. J Hepatol, 51: 581–592.

    Article  CAS  PubMed  Google Scholar 

  • Lin SR, Yang HC, Kuo YT, Liu CJ, Yang TY, Sung KC, Lin YY, Wang HY, Wang CC, Shen YC, Wu FY, Kao JH, Chen DS, Chen PJ. 2014. The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo. Mol Ther Nucleic Acids, 3: e186.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Liu J, Kosinska A, Lu M, Roggendorf M. 2014. New therapeutic vaccination strategies for the treatment of chronic hepatitis B. Virol Sin, 29:10–16.

    Article  PubMed  Google Scholar 

  • Liu X, Hao R, Chen S, Guo D, Chen Y. 2015. Inhibition of Hepatitis B Virus by CRISPR/Cas9 System via Targeting the Conserved Regions of Viral Genome. J Gen Virol.

    Google Scholar 

  • Lucifora J, Xia Y, Reisinger F, Zhang K, Stadler D, Cheng X, Sprinzl MF, Koppensteiner H, Makowska Z, Volz T, Remouchamps C, Chou WM, Thasler WE, Huser N, Durantel D, Liang TJ, Munk C, Heim MH, Browning JL, Dejardin E, Dandri M, Schindler M, Heikenwalder M, Protzer U. 2014. Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science, 343: 1221–1228.

    Article  CAS  PubMed  Google Scholar 

  • Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. 2013. RNA-guided human genome engineering via Cas9. Science, 339: 823–826.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Matthews LA, Simmons LA. 2014. Bacterial nonhomologous end joining requires teamwork. J Bacteriol, 196: 3363–3365.

    Article  PubMed Central  PubMed  Google Scholar 

  • Nassal M. 2015. HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut. pii: gutjnl-2015-309809.

    Google Scholar 

  • Ohno M, Otsuka M, Kishikawa T, Yoshikawa T, Takata A, Koike K. 2015. Novel therapeutic approaches for hepatitis B virus covalently closed circular DNA. World J Gastroenterol, 21: 7084–7088.

    PubMed Central  CAS  PubMed  Google Scholar 

  • Park YM, Jang JW, Yoo SH, Kim SH, Oh IM, Park SJ, Jang YS, Lee SJ. 2014. Combinations of eight key mutations in the X/preC region and genomic activity of hepatitis B virus are associated with hepatocellular carcinoma. J Viral Hepat, 21: 171–177.

    Article  CAS  PubMed  Google Scholar 

  • Perkel J. 2015. CRISPR/Cas faces the bioethics spotlight. Biotechniques, 58: 223–227.

    PubMed  Google Scholar 

  • Ramanan V, Shlomai A, Cox DB, Schwartz RE, Michailidis E, Bhatta A, Scott DA, Zhang F, Rice CM, Bhatia SN. 2015. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. Sci Rep, 5: 10833.

    Article  CAS  PubMed  Google Scholar 

  • Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. 2015. In vivo genome editing using Staphylococcus aureus Cas9. Nature, 520: 186–191.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. 2013. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 154: 1380–1389.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Saayman S, Ali SA, Morris KV, Weinberg MS. 2015. The therapeutic application of CRISPR/Cas9 technologies for HIV. Expert Opin Biol Ther, 15: 819–830.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Schiffer JT, Aubert M, Weber ND, Mintzer E, Stone D, Jerome KR. 2012. Targeted DNA mutagenesis for the cure of chronic viral infections. J Virol, 86: 8920–8936.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Schweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. 2015. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet. pii: S0140-6736(15)61412-X.

    Google Scholar 

  • Seeger C, Mason WS. 2015. Molecular biology of hepatitis B virus infection. Virology, 479–480: 672–686.

    Article  PubMed  Google Scholar 

  • Seeger C, Sohn JA. 2014. Targeting Hepatitis B Virus With CRISPR/Cas9. Mol Ther Nucleic Acids, 3: e216.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sellmyer MA, Chen LC, Egeler EL, Rakhit R, Wandless TJ. 2012. Intracellular context affects levels of a chemically dependent destabilizing domain. PLoS One, 7: e43297.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F. 2014. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science, 343: 84–87.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, Skarnes WC. 2014. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods, 11: 399–402.

    Article  CAS  PubMed  Google Scholar 

  • Smith GR. 2001. Homologous recombination near and far from DNA breaks: alternative roles and contrasting views. Annu Rev Genet, 35: 243–274.

    Article  CAS  PubMed  Google Scholar 

  • Strong CL, Guerra HP, Mathew KR, Roy N, Simpson LR, Schiller MR. 2015. Damaging the Integrated HIV Proviral DNA with TALENs. PLoS One, 10: e0125652.

    Article  PubMed Central  PubMed  Google Scholar 

  • Tang H, Oishi N, Kaneko S, Murakami S. 2006. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Sci, 97: 977–983.

    Article  CAS  PubMed  Google Scholar 

  • Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK. 2014. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol, 32: 569–576.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • van der Ploeg JR. 2009. Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages. Microbiology, 155: 1966–1976.

    Article  PubMed  Google Scholar 

  • Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. 2013. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 153: 910–918.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wang J, Xu ZW, Liu S, Zhang RY, Ding SL, Xie XM, Long L, Chen XM, Zhuang H, Lu FM. 2015. Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication. World J Gastroenterol, 21: 9554–9565.

    Article  PubMed Central  PubMed  Google Scholar 

  • Werle-Lapostolle B, Bowden S, Locarnini S, Wursthorn K, Petersen J, Lau G, Trepo C, Marcellin P, Goodman Z, Delaney WEt, Xiong S, Brosgart CL, Chen SS, Gibbs CS, Zoulim F. 2004. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology, 126: 1750–1758.

    Article  CAS  PubMed  Google Scholar 

  • Wyman C, Kanaar R. 2006. DNA double-strand break repair: all's well that ends well. Annu Rev Genet, 40: 363–383.

    Article  CAS  PubMed  Google Scholar 

  • Xu T, Li Y, Van Nostrand JD, He Z, Zhou J. 2014. Cas9-based tools for targeted genome editing and transcriptional control. Appl Environ Microbiol, 80: 1544–1552.

    Article  PubMed Central  PubMed  Google Scholar 

  • Yan H, Peng B, He W, Zhong G, Qi Y, Ren B, Gao Z, Jing Z, Song M, Xu G, Sui J, Li W. 2013. Molecular determinants of hepatitis B and D virus entry restriction in mouse sodium taurocholate cotransporting polypeptide. J Virol, 87: 7977–7991.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, Huang Y, Qi Y, Peng B, Wang H, Fu L, Song M, Chen P, Gao W, Ren B, Sun Y, Cai T, Feng X, Sui J, Li W. 2014. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife, 3.

    Google Scholar 

  • Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. 2013. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell, 154: 1370–1379.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yang HC, Kao JH. 2014. Persistence of hepatitis B virus covalently closed circular DNA in hepatocytes: molecular mechanisms and clinical significance. Emerg Microbes Infect, 3: e64.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yin H, Xue W, Chen S, Bogorad RL, Benedetti E, Grompe M, Koteliansky V, Sharp PA, Jacks T, Anderson DG. 2014. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol, 32: 551–553.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zhen S, Hua L, Liu YH, Gao LC, Fu J, Wan DY, Dong LH, Song HF, 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.

    Article  CAS  PubMed  Google Scholar 

  • Zhu W, Lei R, Le Duff Y, Li J, Guo F, Wainberg MA, Liang C. 2015. The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA. Retrovirology, 12: 22.

    Article  PubMed Central  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dongliang Yang.

Additional information

ORCID: 0000-0001-5387-2660

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Peng, C., Lu, M. & Yang, D. CRISPR/Cas9-based tools for targeted genome editing and replication control of HBV. Virol. Sin. 30, 317–325 (2015). https://doi.org/10.1007/s12250-015-3660-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12250-015-3660-x

Keywords

Navigation