請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71112完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊宏志(Hung-Chih Yang) | |
| dc.contributor.author | Pei-Chun Hou | en |
| dc.contributor.author | 侯佩君 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:53:36Z | - |
| dc.date.available | 2020-10-09 | |
| dc.date.copyright | 2018-10-09 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-07-30 | |
| dc.identifier.citation | 1. Lavanchy, D., Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. Journal of viral hepatitis, 2004. 11(2): p. 97-107. 2. Thio, C.L., et al., HIV-1, hepatitis B virus, and risk of liver-related mortality in the Multicenter Cohort Study (MACS). The Lancet, 2002. 360(9349): p. 1921-1926. 3. Trépo, C., H.L.Y. Chan, and A. Lok, Hepatitis B virus infection. The Lancet, 2014. 384(9959): p. 2053-2063. 4. Tong, S. and P. Revill, Overview of hepatitis B viral replication and genetic variability. Journal of hepatology, 2016. 64(1): p. S4-S16. 5. Summers, J., A. O'Connell, and I. Millman, Genome of hepatitis B virus: restriction enzyme cleavage and structure of DNA extracted from Dane particles. Proceedings of the National Academy of Sciences, 1975. 72(11): p. 4597-4601. 6. Cattaneo, R., H. Will, and H. Schaller, Hepatitis B virus transcription in the infected liver. The EMBO journal, 1984. 3(9): p. 2191-2196. 7. Su, H. and J.-K. Yee, Regulation of hepatitis B virus gene expression by its two enhancers. Proceedings of the National Academy of Sciences, 1992. 89(7): p. 2708-2712. 8. Koumbi, L. and P. Karayiannis, The Epigenetic Control of Hepatitis B Virus Modulates the Outcome of Infection. Front Microbiol, 2015. 6: p. 1491. 9. Zhang, Y., et al., Comparative analysis of CpG islands among HBV genotypes. PloS one, 2013. 8(2): p. e56711. 10. Zhang, Y., et al., Transcription of hepatitis B virus covalently closed circular DNA is regulated by CpG methylation during chronic infection. PloS one, 2014. 9(10): p. e110442. 11. Milich, D.R., et al., The secreted hepatitis B precore antigen can modulate the immune response to the nucleocapsid: a mechanism for persistence. The Journal of Immunology, 1998. 160(4): p. 2013-2021. 12. Xu, Z., et al., Enhancement of hepatitis B virus replication by its X protein in transgenic mice. Journal of virology, 2002. 76(5): p. 2579-2584. 13. Ni, Y., et al., Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes. Gastroenterology, 2014. 146(4): p. 1070-1083. e6. 14. Yan, H., et al., Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. elife, 2012. 1. 15. Nassal, M., Hepatitis B viruses: reverse transcription a different way. Virus Res, 2008. 134(1-2): p. 235-49. 16. Staprans, S., D. Loeb, and D. Ganem, Mutations affecting hepadnavirus plus-strand DNA synthesis dissociate primer cleavage from translocation and reveal the origin of linear viral DNA. Journal of virology, 1991. 65(3): p. 1255-1262. 17. Yang, W. and J. Summers, Illegitimate replication of linear hepadnavirus DNA through nonhomologous recombination. Journal of virology, 1995. 69(7): p. 4029-4036. 18. Blondot, M.-L., V. Bruss, and M. Kann, Intracellular transport and egress of hepatitis B virus. Journal of hepatology, 2016. 64(1): p. S49-S59. 19. Ott, J.J., et al., Global epidemiology of hepatitis B virus infection: new estimates of age-specific HBsAg seroprevalence and endemicity. Vaccine, 2012. 30(12): p. 2212-2219. 20. Alonso, S., et al., Upcoming pharmacological developments in chronic hepatitis B: can we glimpse a cure on the horizon? BMC Gastroenterol, 2017. 17(1): p. 168. 21. Greenberg, H.B., et al., Effect of human leukocyte interferon on hepatitis B virus infection in patients with chronic active hepatitis. New England Journal of Medicine, 1976. 295(10): p. 517-522. 22. Papatheodoridis, G.V., E. Dimou, and V. Papadimitropoulos, Nucleoside analogues for chronic hepatitis B: antiviral efficacy and viral resistance. The American journal of gastroenterology, 2002. 97(7): p. 1618-1628. 23. Lin, C.L. and J.H. Kao, Review article: novel therapies for hepatitis B virus cure - advances and perspectives. Aliment Pharmacol Ther, 2016. 44(3): p. 213-222. 24. Deveau, H., J.E. Garneau, and S. Moineau, CRISPR/Cas system and its role in phage-bacteria interactions. Annual review of microbiology, 2010. 64: p. 475-493. 25. Makarova, K.S., et al., Evolution and classification of the CRISPR–Cas systems. Nature Reviews Microbiology, 2011. 9(6): p. 467-477. 26. Mojica, F.J., et al., Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology, 2009. 155(3): p. 733-740. 27. Sander, J.D. and J.K. Joung, CRISPR-Cas systems for editing, regulating and targeting genomes. Nature biotechnology, 2014. 32(4): p. 347-355. 28. Jinek, M., et al., RNA-programmed genome editing in human cells. elife, 2013. 2. 29. Cho, S.W., et al., Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nature biotechnology, 2013. 31(3): p. 230-232. 30. Hsu, P.D., E.S. Lander, and F. Zhang, Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014. 157(6): p. 1262-1278. 31. Li, H., et al., Removal of integrated hepatitis B virus DNA using CRISPR-Cas9. Frontiers in cellular and infection microbiology, 2017. 7: p. 91. 32. Lin, S.-R., et al., The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo. Molecular Therapy-Nucleic Acids, 2014. 3. 33. Wang, J., et al., Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication. World journal of gastroenterology: WJG, 2015. 21(32): p. 9554-9565. 34. Zhu, W., et al., CRISPR/Cas9 produces anti-hepatitis B virus effect in hepatoma cells and transgenic mouse. Virus research, 2016. 217: p. 125-132. 35. Ramanan, V., et al., CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. Scientific reports, 2015. 5: p. 10833. 36. Makarova, K.S., et al., An updated evolutionary classification of CRISPR–Cas systems. Nature Reviews Microbiology, 2015. 13(11): p. 722-736. 37. Schunder, E., et al., First indication for a functional CRISPR/Cas system in Francisella tularensis. International Journal of Medical Microbiology, 2013. 303(2): p. 51-60. 38. Vestergaard, G., R.A. Garrett, and S.A. Shah, CRISPR adaptive immune systems of Archaea. RNA biology, 2014. 11(2): p. 156-167. 39. Zetsche, B., et al., Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 2015. 163(3): p. 759-771. 40. Yamano, T., et al., Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell, 2016. 165(4): p. 949-962. 41. Kim, D., et al., Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nature biotechnology, 2016. 34(8): p. 863-868. 42. Kleinstiver, B.P., et al., Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nature biotechnology, 2016. 34(8): p. 869. 43. Fonfara, I., et al., The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature, 2016. 532(7600): p. 517-521. 44. Zetsche, B., et al., Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array. Nature biotechnology, 2017. 35(1): p. 31-34. 45. Zhong, G., et al., Cpf1 proteins excise CRISPR RNAs from mRNA transcripts in mammalian cells. Nature chemical biology, 2017. 13(8): p. 839-841. 46. Li, X., et al., Base editing with a Cpf1–cytidine deaminase fusion. Nature biotechnology, 2018. 36(4): p. 324-327. 47. Zhang, X., et al., Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein cell, 2018. 9(4): p. 380-383. 48. Tak, Y.E., et al., Inducible and multiplex gene regulation using CRISPR–Cpf1-based transcription factors. nature methods, 2017. 14(12): p. 1163-1166. 49. Tu, T., et al., HBV DNA Integration: Molecular mechanisms and clinical implications. Viruses, 2017. 9(4): p. 75. 50. Hai, H., A. Tamori, and N. Kawada, Role of hepatitis B virus DNA integration in human hepatocarcinogenesis. World Journal of Gastroenterology: WJG, 2014. 20(20): p. 6236-6243. 51. Yang, H.-C. and P.-J. Chen, The potential and challenges of CRISPR-Cas in eradication of hepatitis B virus covalently closed circular DNA. Virus research, 2017. 52. Seeger, C., Control of viral transcripts as a concept for future HBV therapies. Current opinion in virology, 2018. 30: p. 18-23. 53. Portela, A. and M. Esteller, Epigenetic modifications and human disease. Nature biotechnology, 2010. 28(10): p. 1057-1068. 54. Gibney, E. and C. Nolan, Epigenetics and gene expression. Heredity, 2010. 105(1): p. 4-13. 55. Vivekanandan, P., D. Thomas, and M. Torbenson, Hepatitis B viral DNA is methylated in liver tissues. Journal of viral hepatitis, 2008. 15(2): p. 103-107. 56. Miller, R.H. and W.S. Robinson, Integrated hepatitis B virus DNA sequences specifying the major viral core polypeptide are methylated in PLC/PRF/5 cells. Proceedings of the National Academy of Sciences, 1983. 80(9): p. 2534-2538. 57. Vivekanandan, P., D. Thomas, and M. Torbenson, Methylation regulates hepatitis B viral protein expression. The Journal of infectious diseases, 2009. 199(9): p. 1286-1291. 58. Vivekanandan, P., et al., Hepatitis B virus replication induces methylation of both host and viral DNA. Journal of virology, 2010. 84(9): p. 4321-4329. 59. Kim, J.-W., et al., Replicative activity of hepatitis B virus is negatively associated with methylation of covalently closed circular DNA in advanced hepatitis B virus infection. Intervirology, 2011. 54(6): p. 316-325. 60. Vojta, A., et al., Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucleic acids research, 2016. 44(12): p. 5615-5628. 61. Park, J., S. Bae, and J.-S. Kim, Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics, 2015. 31(24): p. 4014-4016. 62. Stepper, P., et al., Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase. Nucleic acids research, 2016. 45(4): p. 1703-1713. 63. Siddique, A.N., et al., Targeted methylation and gene silencing of VEGF-A in human cells by using a designed Dnmt3a–Dnmt3L single-chain fusion protein with increased DNA methylation activity. Journal of molecular biology, 2013. 425(3): p. 479-491. 64. O’Geen, H., et al., dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic acids research, 2017. 45(17): p. 9901-9916. 65. Gao, L., et al., Engineered Cpf1 variants with altered PAM specificities. Nature biotechnology, 2017. 35(8): p. 789-792. 66. Huang, Y.-H., et al., DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A. Genome biology, 2017. 18(1): p. 176. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71112 | - |
| dc.description.abstract | B型肝炎病毒（Hepatitis B virus, HBV）感染肝細胞後，其基因體以共價閉合環狀去氧核糖核酸（covalently closed circular DNA, cccDNA）結構或嵌入宿主基因體中存在宿主肝細胞內。嵌入型的B型肝炎基因體（Integrated HBV DNA），並不會再進行複製，但仍可轉錄、轉譯出HBV的表面抗原（HBsAg）。目前治療HBV的藥物- Nucleos(t)ide analogues (NAs)可有效抑制病毒反轉錄複製的過程，但因為無法徹底清除病毒的複製模板-cccDNA，所以仍然無法治癒HBV感染。我們先前的研究以及其他多份文獻中，都證明了HBV的基因體可以被核酸內切酶-Cas9此基因編輯工具破壞；此外，還有另一種核酸內切酶稱作Cpf1，它被分類在第五型CRISPR系統，其特色是具有去氧核醣核酸酶（DNase）及核糖核酸酶（RNase）兩種活性，除了DNA之外，也可以切割RNA，因此只要利用一長條crRNA前驅物（pre-crRNA）就可以切割成數個成熟的crRNA，以引導Cpf1進行多位點專一性編輯，Cpf1會是個適合進行多位點基因編輯的工具。在本篇研究中，我們的目標為利用CRISPR/Cpf1系統對HBV基因體做多位點的基因編輯；我們首先挑選出八條具B型肝炎病毒專一性的crRNA，其引導Cpf1去切割病毒基因體並誘發基因的插入或刪除（Indels）；接著我們將數個crRNA組成pre-crRNA，在體外測試其基因編輯能力，結果能同時切割三個HBV CpG island，使B型肝炎的核心抗原（HBc），e抗原（HBe）、表面抗原（HBs）同時下降。我們也更進一步發展出以CRISPR/Cpf1為基礎的工具，其由無DNase活性的LbCpf1（簡稱dCpf1）及DNA甲基轉移酶（DNA methyltransferase）所組成，目的是希望能將HBV的基因體進行位點專一性的甲基化，利用此非切斷基因的方法來抑制HBV的基因表現。我們認為，多位點的甲基化可以更有效且專一的去抑制HBV的基因表現。本篇研究成功利用CRISPR/Cpf1系統對HBV基因體進行多位點基因編輯，在體外能有效破壞B型肝炎病毒基因體，以降低其表現，將來有潛力做為根除B型肝炎病毒之抗病毒藥物。 | zh_TW |
| dc.description.abstract | Following the entry of hepatitis B virus (HBV) into hepatocytes, a minor portion of viral DNAs integrate into host chromosomes. Although the integrated HBV genome is not required for HBV replication, it can continuously produce HBV surface antigens, and is found in 85%-90% of HBV-related HCCs. Nucleos(t)ide analogues (NAs) inhibit the reverse transcription of HBV replication cycle very effectively, but they cannot cure HBV infection because they fail to eliminate the replicative template covalently closed circular DNA (cccDNA) and the integrated HBV genomes, which can still produce viral antigens. Previous studies, including ours, have shown that the HBV genome can be disrupted by the RNA-guided endonuclease Cas9. Cpf1 nuclease belongs to the type V CRISPR system and has dual DNase and RNase activities, so it can produce multiple CRISPR RNAs (crRNAs) from a single long RNA (pre-crRNA). Therefore, Cpf1 is an ideal tool for multiplex gene editing. In this studies, we aimed to utilize the CRISPR/Cpf1 system to target HBV DNA. We first screened an array of HBV-specific protospacer sequences and found eight of them were able to induce indel formation by the CRISPR/Cpf1 system. Furthermore, we used Cpf1 and multiple HBV-specific crRNAs to cut on three CpG islands and simultaneously reduced HBc, HBe, and HBs antigens. Furthermore, we developed the CRISPR/Cpf1-based tool, consisting of DNase inactive LbCpf1(dLbCpf1) and a catalytic domain of DNA methyltransferase DNMT3A, to methylate HBV genome in a site-specific manner for the reduction of gene expression without cleavage. We hypothesize that multiplex methylation can suppress HBV gene expression more specifically and efficiently. In conclusion, we demonstrated the ability and potential of the CRISPR/Cpf1 system for multiplex targeting HBV genome in the clearance of HBV in chronic hepatitis B patients. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:53:36Z (GMT). No. of bitstreams: 1 ntu-107-R05445105-1.pdf: 2305232 bytes, checksum: 8542cae965c4c02de0e36bed6306288c (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員審定書 i 致謝 ii 中文摘要 iii 英文摘要 v 1 Introduction 1 1.1 Epidemiology of HBV infection 1 1.2 Genome structure and organization of HBV 1 1.3 Functions of HBV proteins 2 1.4 The replication cycle of HBV 3 1.5 Limitations of current treatment for chronic hepatitis B 5 1.6 Application of the genome editing tool - CRISPR/Cas to HBV 6 1.6.1 Introduction of CRISPR/Cas technology 6 1.6.2 Disruption of HBV genome by the CRISPR/Cas9 system 7 1.7 The property of CRISPR/Cpf1 for multiplex genome editing 8 1.7.1 Introduction of CRISPR/Cpf1 8 1.7.2 Dual RNA/DNA nuclease functions of Cpf1: pre-crRNA processing 9 1.8 Concerns and solutions of the CRISPR/Cas system targeting HBV 10 1.8.1 The risk of cutting HBV DNA by Cas-associated nucleases 10 1.8.2 HBV DNA methylation and viral expression 11 1.8.3 The potential role of CRISPR/Cas-based epigenetic modulators regulating HBV gene expression 11 2 Specific Aims 13 3 Materials and Methods 14 3.1 Plasmids 14 3.2 HBV-specific crRNA design and cloning 15 3.3 Pre-crRNA array design and cloning 16 3.4 Cell lines and culture conditions 17 3.5 DNA transfection 17 3.6 Lentiviral transduction of HepG2.2.15 18 3.7 Analysis of DNA methylation 19 3.8 Analysis of extracellular and intracellular HBV proteins 20 3.9 Analysis of HBV genome cleavage by NGS 22 4 Results 23 4.1 Co-expression of HBV-specific crRNAs and Cpf1 down-regulates the production of HBV proteins 23 4.2 Multiplex cleavage of HBV genome by Cpf1/pre-crRNA array decreases HBV expression 24 4.3 Cleavage of multiple CpG islands reduces the expression of individual HBV proteins 25 4.4 dLbCpf1-DNMT3A.DNMT3L induces targeted CpG methylation at the BACH2 locus of HEK293T cells 26 4.5 The application of pdLbCpf1-DNMT3A.DNMT3L to site-specifically increase the methylation level of HBV genome in HepG2.2.15 28 4.6 Optimization of the Cpf1-based genome editing tool 28 5 Discussion 30 5.1 Utility of CRISPR/Cpf1 in the treatment of HBV infection 30 5.2 Multiplex disruption of HBV genome by using simplified CRISPR/pre-crRNA systems 30 5.3 Development of Cpf1-based tools for site-specific DNA methylation 31 5.4 The application of CRISPR-Cpf1-based DNA methyltransferase in HBV DNA methylation 32 6 Figures 34 Figure 1. Designs of crRNAs targeting HBV genome. 34 Figure 2. The efficacy of HBV-specific crRNAs/LbCpf1 on disrupting HBV genome. 35 Figure 3. The efficacy of multiplexed HBV specific crRNAs/LbCpf1 on regulating the HBV expression. 36 Figure 4. Multiplex targeting of all HBV CpG island simultaneously via pre-crRNA/Cpf1. 38 Figure 5. Targeted CpG methylation of the BACH2 gene via dLbCpf1-DNMT3A-DNMT3L tool in HEK293T cells. 40 Figure 6. Targeting dLbCpf1-DNMT3A-DNMT3L to HBV CpG islands. 42 Figure 7. Modification of Cpf1-based methylation modulator. 44 7 References 46 8 Supplementary Information 51 Table 1. List of the HBV-specific single Cpf1 crRNA 51 Table 2. DNA oligonucleotides for pre-crRNA array cloning 52 Table 3. PCR primers and pyrosequencing primers 53 Table 4. Primers for the first round PCR amplification before deep sequencing 53 Figure S1. 54 | |
| dc.language.iso | en | |
| dc.subject | B 型肝炎病毒 | zh_TW |
| dc.subject | 基因體甲基化 | zh_TW |
| dc.subject | Cpf1 | zh_TW |
| dc.subject | CRISPR | zh_TW |
| dc.subject | 基因編輯 | zh_TW |
| dc.subject | 嵌入型 B 型肝炎基因體 | zh_TW |
| dc.subject | integrated HBV DNA | en |
| dc.subject | Hepatitis B virus | en |
| dc.subject | DNA methylation | en |
| dc.subject | Cpf1 | en |
| dc.subject | CRISPR | en |
| dc.subject | gene editing | en |
| dc.title | 利用 CRISPR/Cpf1 系統對 B 型肝炎病毒進行多位點基因編輯 | zh_TW |
| dc.title | Multiplex targeting HBV via CRISPR/Cpf1 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳佑宗(You-Tzung Chen),董馨蓮(Shin-Lian Doong),凌嘉鴻(Steven Lin) | |
| dc.subject.keyword | B 型肝炎病毒,嵌入型 B 型肝炎基因體,基因編輯,CRISPR,Cpf1,基因體甲基化, | zh_TW |
| dc.subject.keyword | Hepatitis B virus,integrated HBV DNA,gene editing,CRISPR,Cpf1,DNA methylation, | en |
| dc.relation.page | 54 | |
| dc.identifier.doi | 10.6342/NTU201802152 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-07-30 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-107-1.pdf 未授權公開取用 | 2.25 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。