CRISPR/Cas9 技术在乙型肝炎病毒基因组抑制中的应用_West China Medical Journal

Authors：

 唐光敏 , 周威龙

四川大学华西医院感染性疾病中心（成都 610041）;

Corresponding author：

唐光敏, Email: 52930382@qq.com

Keywords：

CRISPR/Cas9; 乙型肝炎病毒; 基因组抑制; 基因编辑

DOI：

10.7507/1002-0179.201606030

Video：

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目前世界范围内约有 2.4 亿慢性乙型肝炎病毒（hepatitis B virus，HBV）感染者，HBV 感染是世界性的重大公共卫生难题。随着分子生物学工具的不断发展，目前第 3 代基因定点编辑技术 CRISPR/Cas9 作为热点已经广泛地应用于多种病毒的研究与实验性治疗中。该文简要回顾了 HBV 基因组的特点、基因编辑技术的发展及原理和 CRISPR/Cas9 在 HBV 基因组抑制中的研究现状及局限性。相对于锌指核糖核酸酶和转录激活因子样效应物核酸酶其他两种基因编辑技术，CRISPR/Cas9 技术极大地提高了基因编辑的能力。虽然目前仍属于概念证明阶段，但多数基础研究均证实了 CRISPR/Cas9 技术在体内外对 HBV 基因组具有编辑能力并能降低其 DNA 复制与病毒蛋白的表达能力。在潜在安全风险及基因编辑载体的输送效率等问题得到解决后，CRISPR/Cas9 技术联合逆转录抑制药物的治疗将为 HBV 感染的临床治愈带来曙光。

Citation： 唐光敏, 周威龙. CRISPR/Cas9 技术在乙型肝炎病毒基因组抑制中的应用. West China Medical Journal, 2017, 32(12): 1967-1971. doi: 10.7507/1002-0179.201606030 Copy

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2.	WHO Guidelines Approved by the Guidelines Review Committee. Guidelines for the prevention, care and treatment of persons with chronic hepatitis B infection. 2015. https://www.ncbi.nlm.nih. gov/pubmed/26225396.
3.	Pingoud A, Wilson GG, Wende W. Type II restriction endonucleases-a historical perspective and more. Nucleic Acids Res, 2014, 42(12): 7489-7527.
4.	Gaj T, Gersbach CA, Barbas CF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol, 2013, 31(7): 397-405.
5.	Klug A. The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annu Rev biochem, 2010, 79: 213-231.
6.	Christian M, Cermak T, Doyle EL, et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics, 2010, 186(2): 757-761.
7.	Wasik BR, Turner PE. On the biological success of viruses. Annu Rev microbiol, 2013, 67: 519-541.
8.	Grissa I, Vergnaud G, Pourcel C. Clustered regularly interspaced short palindromic repeats (CRISPRs) for the genotyping of bacterial pathogens//Molecular Epidemiology of Microorganisms. Humana Press, 2009: 105-116.
9.	Swarts DC, Mosterd C, van Passel MW, et al. CRISPR interference directs strand specific spacer acquisition. PLoS One, 2012, 7(4): e35888.
10.	Yosef I, Goren MG, Qimron U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res, 2012, 40(12): 5569-5576.
11.	Nuñez JK, Kranzusch PJ, Noeske J, et al. Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity. Nat Struct Mol Biol, 2014, 21(6): 528-534.
12.	Nuñez JK, Lee AS, Engelman A, et al. Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity. Nature, 2015, 519(7542): 193-198.
13.	Barrangou R, Marraffini LA. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Mol Cell, 2014, 54(2): 234-244.
14.	Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121): 819-823.
15.	Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014, 157(6): 1262-1278.
16.	Hsu PD, Scott DA, Weinstein JA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 2013, 31(9): 827-832.
17.	Mali P, Yang LH, Esvelt KM, et al. RNA-Guided human genome engineering via Cas9. Science, 2013, 339(6121): 823-826.
18.	Garneau JE, Dupuis MÈ, Villion M, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 2010, 468(7320): 67-71.
19.	Gasiunas G, Barrangou R, Horvath PA. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A, 2012, 109(39): E2579-E2586.
20.	Qi LS, Larson MH, Gilbert LA, et al. Repurposing CRISPR as an RNA-Guided platform for Sequence-Specific control of gene expression. Cell, 2013, 152(5): 1173-1183.
21.	Lin SR, Yang HC, Kuo YT, et al. The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo. Mol Ther Nucleic Acids, 2014, 3: e186.
22.	Kennedy EM, Bassit LC, Mueller HA, et al. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease. Virology, 2015, 476: 196-205.
23.	Liu X, Hao R, Chen S, et al. Inhibition of hepatitis B virus by the CRISPR/Cas9 system via targeting the conserved regions of the viral genome. J Gen Virol, 2015, 96(8): 2252-2261.
24.	Zhen S, Hua L, Liu YH, et al. Harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated Cas9 system to disrupt the hepatitis B virus. Gene Ther, 2015, 22(5): 404-412.
25.	Dong C, Qu L, Wang H, et al. Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication. Antiviral Res, 2015, 118: 110-117.
26.	Ramanan V, Shlomai A, Cox DB, et al. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. Sci Rep, 2015, 5: 10833.
27.	Karimova M, Beschorner N, Dammermann W, et al. CRISPR/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X. Sci Rep, 2015, 5: 13734.
28.	Wang J, Xu ZW, Liu S, et al. Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication. World J Gastroenterol, 2015, 21(32): 9554-9565.
29.	Dejean A, Lugassy C, Zafrani S, et al. Detection of hepatitis B virus DNA in pancreas, kidney and skin of two human carriers of the virus. J Gen Virol, 1984, 65(Pt3): 651-655.
30.	Mason A, Wick M, White H, et al. Hepatitis B virus replication in diverse cell types during chronic hepatitis B virus infection. Hepatology, 1993, 18(4): 781-789.
31.	Pontisso P, Poon MC, Tiollais P, et al. Detection of hepatitis B virus DNA in mononuclear blood cells. Br Med J(Clin Res Ed), 1984, 288(6430): 1563-1566.
32.	Ran FA, Cong L, Yan WX, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature, 2015, 520(7546): 186-191.
33.	Truong DJ, Küehner K, Küehn R, et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res, 2015, 43(13): 6450-6458.
34.	Fu Y, Foden JA, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol, 2013, 31(9): 822-826.
35.	Mali P, Aach J, Stranges PB, et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol, 2013, 31(9): 833-838.
36.	Fu YF, Sander JD, Reyon D, et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol, 2014, 32(3): 279-284.
37.	Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokⅠ nuclease improves the specificity of genome modification. Nat Biotechnol, 2014, 32(6): 577-582.
38.	Feitelson MA, Lee J. Hepatitis B virus integration, fragile sites, and hepatocarcinogenesis. Cancer Lett, 2007, 252(2): 157-170.

1. Ott JJ, Stevens GA, Groeger J, et al. Global epidemiology of hepatitis B virus infection: New estimates of age-specific HBsAg seroprevalence and endemicity. Vaccine, 2012, 30(12): 2212-2219.
2. WHO Guidelines Approved by the Guidelines Review Committee. Guidelines for the prevention, care and treatment of persons with chronic hepatitis B infection. 2015. https://www.ncbi.nlm.nih. gov/pubmed/26225396.
3. Pingoud A, Wilson GG, Wende W. Type II restriction endonucleases-a historical perspective and more. Nucleic Acids Res, 2014, 42(12): 7489-7527.
4. Gaj T, Gersbach CA, Barbas CF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol, 2013, 31(7): 397-405.
5. Klug A. The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annu Rev biochem, 2010, 79: 213-231.
6. Christian M, Cermak T, Doyle EL, et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics, 2010, 186(2): 757-761.
7. Wasik BR, Turner PE. On the biological success of viruses. Annu Rev microbiol, 2013, 67: 519-541.
8. Grissa I, Vergnaud G, Pourcel C. Clustered regularly interspaced short palindromic repeats (CRISPRs) for the genotyping of bacterial pathogens//Molecular Epidemiology of Microorganisms. Humana Press, 2009: 105-116.
9. Swarts DC, Mosterd C, van Passel MW, et al. CRISPR interference directs strand specific spacer acquisition. PLoS One, 2012, 7(4): e35888.
10. Yosef I, Goren MG, Qimron U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res, 2012, 40(12): 5569-5576.
11. Nuñez JK, Kranzusch PJ, Noeske J, et al. Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity. Nat Struct Mol Biol, 2014, 21(6): 528-534.
12. Nuñez JK, Lee AS, Engelman A, et al. Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity. Nature, 2015, 519(7542): 193-198.
13. Barrangou R, Marraffini LA. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Mol Cell, 2014, 54(2): 234-244.
14. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121): 819-823.
15. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014, 157(6): 1262-1278.
16. Hsu PD, Scott DA, Weinstein JA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 2013, 31(9): 827-832.
17. Mali P, Yang LH, Esvelt KM, et al. RNA-Guided human genome engineering via Cas9. Science, 2013, 339(6121): 823-826.
18. Garneau JE, Dupuis MÈ, Villion M, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 2010, 468(7320): 67-71.
19. Gasiunas G, Barrangou R, Horvath PA. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A, 2012, 109(39): E2579-E2586.
20. Qi LS, Larson MH, Gilbert LA, et al. Repurposing CRISPR as an RNA-Guided platform for Sequence-Specific control of gene expression. Cell, 2013, 152(5): 1173-1183.
21. Lin SR, Yang HC, Kuo YT, et al. The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo. Mol Ther Nucleic Acids, 2014, 3: e186.
22. Kennedy EM, Bassit LC, Mueller HA, et al. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease. Virology, 2015, 476: 196-205.
23. Liu X, Hao R, Chen S, et al. Inhibition of hepatitis B virus by the CRISPR/Cas9 system via targeting the conserved regions of the viral genome. J Gen Virol, 2015, 96(8): 2252-2261.
24. Zhen S, Hua L, Liu YH, et al. Harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated Cas9 system to disrupt the hepatitis B virus. Gene Ther, 2015, 22(5): 404-412.
25. Dong C, Qu L, Wang H, et al. Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication. Antiviral Res, 2015, 118: 110-117.
26. Ramanan V, Shlomai A, Cox DB, et al. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. Sci Rep, 2015, 5: 10833.
27. Karimova M, Beschorner N, Dammermann W, et al. CRISPR/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X. Sci Rep, 2015, 5: 13734.
28. Wang J, Xu ZW, Liu S, et al. Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication. World J Gastroenterol, 2015, 21(32): 9554-9565.
29. Dejean A, Lugassy C, Zafrani S, et al. Detection of hepatitis B virus DNA in pancreas, kidney and skin of two human carriers of the virus. J Gen Virol, 1984, 65(Pt3): 651-655.
30. Mason A, Wick M, White H, et al. Hepatitis B virus replication in diverse cell types during chronic hepatitis B virus infection. Hepatology, 1993, 18(4): 781-789.
31. Pontisso P, Poon MC, Tiollais P, et al. Detection of hepatitis B virus DNA in mononuclear blood cells. Br Med J(Clin Res Ed), 1984, 288(6430): 1563-1566.
32. Ran FA, Cong L, Yan WX, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature, 2015, 520(7546): 186-191.
33. Truong DJ, Küehner K, Küehn R, et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res, 2015, 43(13): 6450-6458.
34. Fu Y, Foden JA, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol, 2013, 31(9): 822-826.
35. Mali P, Aach J, Stranges PB, et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol, 2013, 31(9): 833-838.
36. Fu YF, Sander JD, Reyon D, et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol, 2014, 32(3): 279-284.
37. Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokⅠ nuclease improves the specificity of genome modification. Nat Biotechnol, 2014, 32(6): 577-582.
38. Feitelson MA, Lee J. Hepatitis B virus integration, fragile sites, and hepatocarcinogenesis. Cancer Lett, 2007, 252(2): 157-170.

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West China Medical Journal

CRISPR/Cas9 技术在乙型肝炎病毒基因组抑制中的应用

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