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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳培哲(Pei-Jer Chen) | |
| dc.contributor.author | Chia-Wen Chang | en |
| dc.contributor.author | 張嘉雯 | zh_TW |
| dc.date.accessioned | 2023-03-19T22:52:57Z | - |
| dc.date.copyright | 2022-10-03 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-08-01 | |
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Crispr j, 2019. 2(4): p. 223-229. 31. Ladner, S.K., et al., Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother, 1997. 41(8): p. 1715-20. 32. Farrell, G.C., Clinical Potential of Emerging New Agents in Hepatitis B. Drugs, 2000. 60(4): p. 701-710. 33. Bock, C.T., et al., Structural organization of the hepatitis B virus minichromosome. J Mol Biol, 2001. 307(1): p. 183-96. 34. Chong, C.K., et al., Role of hepatitis B core protein in HBV transcription and recruitment of histone acetyltransferases to cccDNA minichromosome. Antiviral Res, 2017. 144: p. 1-7. 35. Komor, A.C., et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 2016. 533(7603): p. 420-424. 36. Kalle, E., M. Kubista, and C. Rensing, Multi-template polymerase chain reaction. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/85250 | - |
| dc.description.abstract | B型肝炎病毒感染目前為無法根治的疾病之一，在2019年全球已超過2億人口帶有慢性B型肝炎，並且慢性B型肝炎可能會導致肝癌的發生，故慢性B型肝炎病毒感染在全世界上是一個重要的公共衛生議題。目前臨床用的B型肝炎治療藥物雖可以抑制HBV的產生，但因目前使用的藥物無法根除HBV cccDNA，一旦中斷治療，潛伏的HBV cccDNA就能轉錄及轉譯出HBV的RNA及蛋白質並生成更多HBV。故要根治B型肝炎，必須以清除HBV cccDNA為主要目標。近期研究以CRISPR-Cas基因編輯系統作為工具來破壞HBV cccDNA，但CRISPR-Cas的缺點為會造成雙股DNA的斷裂，若是CRISPR-Cas剪切到鑲嵌在宿主染色體中的HBV DNA有可能會造成宿主DNA斷裂而損傷。為了增加CRISPR-Cas的安全性，我們選擇了不會造成雙股DNA斷裂，而是只會對鹼基進行編輯的CRISPR-Cas衍生物的單鹼基編輯器 – ABE。雖然ABE不會對宿主染色體造成損害，但仍有編輯到非目標序列的風險，進而造成宿主染色體的點突變，所以提升ABE對於編輯HBV cccDNA的專一性為本篇主要研究目的。本篇研究中，我們設計了ABE在HBV DNA的編輯位置，在原ABE的C端接上HBc蛋白第151至183的氨基酸，並利用HepAD38細胞株作為分析ABE編輯效率的細胞模式測定編輯效率及專一性是否提升。我們的結果顯示大部分編輯效果都低於10%，雖成功建立融合蛋白，但並未提升目標序列的編輯效率。 | zh_TW |
| dc.description.abstract | Chronic HBV infection is the leading cause of liver cirrhosis and hepatocellular carcinoma. In 2019, the patients with CHB are more than 200 million, and there is no cure for completely elimination of HBV from patients. The main reason that no cure for CHB is the persistence of HBV cccDNA. As the treatment is discontinuous, the existed cccDNA would transcribe and translate HBV RNA and protein, respectively. The transcription and translation of HBV RNA and protein would further release more HBV to patients’ blood. In order to clear HBV cccDNA, CRISPR-Cas technology has been used recently. However, CRISPR-Cas may generate DSBs in host chromosome when targeting to integrated HBV DNA and further induce host DNA damage. For the safety of host, we chose one of the base editors – ABE which converts the base of DNA from A to G without inducing DSB in DNA. Although ABE does not induce DSBs, ABE might have off-target effects and generate point mutation in host chromosome. Hence, enhancing the editing specificity and efficiency of ABE on HBV cccDNA is the main goal of our research. In this study, we first screened and chose the gRNAs that target on HBV DNA, and then engineered ABE by fusing C terminal domain of HBc to C terminus of ABE, and applied ABE and gRNAs in HepAD38 cell model. The results indicated that although we successfully engineered the HBcCTD to ABE, the base editing efficiency was not enhanced. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T22:52:57Z (GMT). No. of bitstreams: 1 U0001-0108202215213500.pdf: 9644252 bytes, checksum: 1eb2dc8141cab50930a7dd286efe720f (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 口試委員審定書 i 謝辭 ii 中文摘要 iii ABSTRACT iv CONTENTS vi CHAPTER 1: INTRODUCTION 1 1.1. Hepatitis B virus 1 1.1.1. General information 1 1.1.2. Life cycle of HBV infection 1 1.1.3. Chronic HBV infection 4 1.1.4. The cure for chronic HBV infection 5 1.2. Gene editing tools 6 1.2.1. CRISPR-Cas system 6 1.2.1.1. General background of CRISPR-Cas system 6 1.2.1.2. The mechanism of CRISPR-Cas system on gene editing 8 1.2.1.3. Application of CRISPR-Cas system against HIV and HBV infection 11 1.2.2. Base editors 12 1.2.2.1. Introduction of base editors 12 1.2.2.2. ABE8e-NRCH 14 1.2.2.3. Application of BEs on viruses 15 1.2.3. Engineering CRISPR-Cas to enhance specificity 16 1.2.3.1. Fusing DNA binding domain with CRISPR-Cas system 16 1.3. Hypothesis 17 CHAPTER 2: MATERIAL AND METHODS 18 2.1. Cell culture 18 2.1.1. HepG2-AD38 18 2.2. Plasmid constructs 18 2.2.1. pCMV-ABE8e-NRCH 18 2.2.2. pLenti-U6-gRNA-BSD 19 2.2.3. pCMV-ABE8e-NRCH-GGS-HBcCTD and pCMV-ABE8e-NRCH-XTEN-HBcCTD 19 2.2.4. pEGFP-N1 20 2.3. Base editing 20 2.4. Southern blot 22 2.5. Western blot 23 CHAPTER 3: RESULTS 25 3.1. Selecting HepG2-AD38 cell model for testing efficiency of ABE 25 3.1.1. HBV DNA species in HepAD38 cells 25 3.1.2. Time course of HBV DNA expression in HepAD38 26 3.1.3. Enhancement of HBV cccDNA majority by ETV treatment in HepAD38 27 3.2. Designing gRNA for ABE to edit HBV DNA 29 3.2.1. Screening appropriate target sequence by multiple sequence alignment of HBV genotypes 29 3.2.2. Test editing efficiency of gRNA 2, 5, 7 on integrated and episomal HBV DNA 30 3.2.3. Evaluation of editing efficiency of gRNA 2 after HBV replication 31 3.3. Enhancing editing efficiency by improving transfection efficiency 32 3.3.1. Adjustment of transfection condition 32 3.3.2 Transfection of ABE and gRNA by electroporation 33 3.4. Enhancing the efficiency and specificity of ABE to HBV cccDNA by fusing C-terminal domain of HBc 34 3.4.1. Establishment of ABE-HBcCTD fusion protein expressing plasmid 34 3.4.2. Evaluation of the base editing efficiency of fusion protein 35 CHAPTER 4: DISCUSSIONS 37 CHAPTER 5: REFERENCES 42 CHAPTER 6: FIGURES 46 Figure 6.1. HBV DNA expressed in HepAD38 in the absence of DOXY. 46 Figure 6.2. Schematic illustration of HBV DNA distribution under DOXY control. 47 Figure 6.3. Time course of the generation of HBV DNA variants in HepAD38. 50 Figure 6.4. The changes of HBV DNA after the different time point treatment of ETV. 53 Figure 6.5. Candidate gRNAs for ABE editing HBV DNA. 55 Figure 6.6. Transfection efficiency and base editing efficiency of selected gRNAs. 57 Figure 6.7. The change of editing ratio after HBV expression in HepAD38. 58 Figure 6.8. The enhancing transfection efficiency and base editing efficiency by conducting transfection twice. 60 Figure 6.9. The changing of base editing efficiency by transfecting different molar ratio of ABE and gRNA2. 62 Figure 6.10. Delivery of ABE and gRNA2 by electroporation. 64 Figure 6.11. The construction of ABE8e-NRCH-HBcCTD fusion proteins expressing plasmid. 66 Figure 6.12. The base editing efficiency of ABE8e-NRCH-HBcCTD fusion proteins. 67 CHAPTER 7: TABLES 68 Table 7.1. The primer sets used to generate inserts for the cloning of fusion proteins. 68 Table 7.2. The primer sets for amplifying gRNA editing target sites. 69 Table 7.3. The primer sets used for the production of HBx probe. 70 Table 7.4. Candidate gRNAs. 71 Table 7.5. The parameters of electroporation. 72 CHAPTER 8: APPENDIX 73 8.1. Life cycle of HBV 73 8.2. Plasmid map – pCMV-ABE8e-NRCH 74 8.3. Plasmid map – pLenti-U6-gRNA-BSD 75 8.4. Plasmid map – pCMV-ABE8e-NRCH-GGS-HBcCTD 76 8.5. Plasmid map – pCMV-ABE8e-NRCH-XTEN-HBcCTD 77 8.6. Plasmid map – pEGFP-N1 78 | |
| dc.language.iso | en | |
| dc.title | 探討單鹼基編輯器的改造以提升其對於B型肝炎病毒之共價閉合環狀去氧核醣核酸的編輯效率及專一性 | zh_TW |
| dc.title | Enhancing the Efficiency and Specificity of Adenine Base Editor on Hepatitis B Virus Covalently Closed Circular DNA by Engineering Editor Components | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊宏志(Hung-Chi Yang),凌嘉鴻(Steven Lin) | |
| dc.subject.keyword | B型肝炎病毒,共價閉合環狀去氧核醣核酸,單鹼基編輯器,單鹼基編輯器融合蛋白, | zh_TW |
| dc.subject.keyword | HBV,cccDNA,ABE,ABE fusion protein, | en |
| dc.relation.page | 78 | |
| dc.identifier.doi | 10.6342/NTU202201935 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-08-01 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2022-10-03 | - |
| 顯示於系所單位： | 微生物學科所 | |
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