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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 | Yu-Chan Yang | en |
dc.contributor.author | 楊于嬋 | zh_TW |
dc.date.accessioned | 2021-07-11T14:55:51Z | - |
dc.date.available | 2021-09-01 | |
dc.date.copyright | 2020-09-04 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-04-10 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78416 | - |
dc.description.abstract | 目前的抗病毒藥物無法根治慢性B型肝炎(chronic hepatitis B, CHB)的主要障礙,是因為受感染的肝臟細胞內存在著B型肝炎病毒(hepatitis B virus, HBV)的複製模板共價閉合環狀去氧核糖核酸(covalently closed circular DNA, cccDNA),以潛在病毒庫持續存在。理論上,要達到完全根治CHB,需要在不破壞宿主細胞基因前提下,消滅所有HBV的基因,包含了HBV cccDNA和鑲嵌入宿主細胞的嵌入型去氧核醣核酸(integrated DNA)。近年來,很多研究證實CRISPR/Cas9基因編輯系統透過專一性破壞HBV基因體,可以作為治療CHB的潛力工具。然而,CRISPR/Cas9基因編輯系統不只會破壞cccDNA,還可以標的作用在鑲嵌在我們染色體上的HBV基因體而誘發雙股去氧核醣核酸的斷裂(double strand breaks, DSBs),導致宿主基因體的重新排列或是損傷的產生。為了增加CRISPR/Cas9基因編輯系統使用上的安全性與應用性,我們使用非切割式CRISPR/Cas9衍伸之基因編輯系統,在不造成DSBs去活化HBV cccDNA和integrated HBV DNA。首先,我們應用SpCas9取得的單鹼基編輯器(Base editor; BE)造成無義突變(nonsense mutation)去活化HBV 基因體。透過篩選取得能造成無義突變的gRNAs。接著使用已帶有HBV基因體的細胞株,將SpCas9-BE和gRNAs一起送入細胞後,觀察到HBV基因體能有效地被編輯,且HBV的聚合酶(polymerase)和表面蛋白都有效地被抑制,有些gRNAs標地的位置有一石二鳥的作用,具有同時抑制HBV聚合酶和表面蛋白的功能。此外,我們也成功的在HBV細胞感染系統中,透過SpCas9-BE作用在游離基因的(episomal)cccDNA能有效的抑制病毒基因的表現。目前正在小鼠模式中實驗,運用腺病毒為基礎的intein調節分裂Cas9單鹼基編輯系統(adeno-associated virus (AAV)-based intein-mediated split-Cas9-BE delivery system)編輯HBV基因體。除此之外,我們也在發展其他非切割式CRISPR/Cas9衍伸之基因編輯系統來抑制HBV基因的表現,像是透過表觀遺傳學(epigenetic)調控HBV基因體的表現,或是運用最新的先導編輯(prime editing)工具,它將失活Cas9接上遺傳工程反轉錄酶(reverse transcriptase)和prime editing guide RNA(pegRNA)作用在HBV啟動子的部分,抑制病毒蛋白的表現,或是使用前間隔序列鄰近構形(protospacer adjacent motif; PAM)限制較小的SpCas9-NG,尋找適合的gRNAs可以作用在不同基因型上的HBV。最後,希望透過非切割式CRISPR/Cas9衍伸之基因編輯系統對於HBV基因體造成永久失去活性能成功,則最終可能可以根治HBV的感染。 | zh_TW |
dc.description.abstract | Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is a major barrier to a cure of chronic hepatitis B (CHB) by current antiviral therapy. In theory, to cure chronic hepatitis B, it is required to eliminate all the replication-competent HBV DNAs without damaging the host genomic DNAs. Recently, it has been shown that the CRISPR/Cas9 system can be utilized for site-specific cleavage of HBV DNA and bears the potential to cure CHB. However, because cccDNA and integrated HBV DNA share almost the same DNA sequences, the CRISPR/Cas system inevitably targets integrated HBV DNA and induces double-strand breaks (DSBs) of host genome, which bear the risk of genomic rearrangement and damage. To enhance the safety and applicability of the CRISPR/Cas9 system in treating CHB, an ideal strategy with CRISPR/Cas9 needs to effectively inactivate HBV genomes without induction of DSBs of host genomes. To achieve this goal, we examined the utility of recently developed CRISPR/Cas-mediated non-cleavage strategies in inactivating HBV genomes, including base editors (BEs) and DNA methylation. We first adopted CRISPR-BE to determine its use in inactivation of HBV genomes. Candidate target sites of the SpCas9-derived base editors (BE) and its variants in HBV genomes were screened for generating nonsense mutations of viral genes with individual guide RNAs (gRNAs). SpCas9-BE with certain gRNAs effectively base-edited polymerase and surface genes and reduced HBV gene expression in cells harboring integrated HBV genomes, but induced very few indels. Some point mutations introduced by base editing resulted in simultaneous suppression of both polymerase and surface genes. Interestingly, we demonstrated that the episomal cccDNA could be successfully edited by SpCas9-BE for suppression of viral gene expression in an in vitro HBV infection system, although the efficacy still needs to be improved. In addition, we also evaluated the utility of the CRISPR-methyltransferase system to silence HBV genome. DNA methylation is an epigenetic mechanism that regulates gene expression. We took advantage of the CRISPR/Cas9-mediated DNA methyltransferase system to introduce de novo methylation of HBV genomes that may suppress viral gene expression. Finally, we examined whether the recombinant AAV delivery of intein-mediated split-Cas9 could be utilized for HBV genome editing in vivo. In summary, we demonstrated that Cas9-mediated non-cleavage approach, particularly Cas9-BE system, it can be utilized for permanent inactivation of cccDNA and integrated HBV DNA without DSBs of host genome, providing a potential safe strategy to cure CHB. Nevertheless, there remain several challenging issues that need to be solved before the realization of CRISPR/Cas9-mediated non-cleavage strategies for HBV cure. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T14:55:51Z (GMT). No. of bitstreams: 1 ntu-109-D00445003-1.pdf: 7727629 bytes, checksum: dac95ab78de2b124895dd656363d6522 (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 中文摘要...................................................................................I Abstract……………………………………………………………………………... II Table of contents…………………………………………………...……………III Introduction 1.1 HBV epidemiology and public health…………………….........1 1.2 Virion structure and genome of HBV…………………….………2 1.3 HBV life cycle……………………………………….……….…..………..4 1.4 HBV DNA integration…………………………….……………………..5 1.5 Integrated HBV DNA and cccDNA are two major obstacles for eradicating HBV from CHB patients………………………………………7 1.6 Genome-editing tools…………………………………..……………...8 1.7 CRISPR/Cas9 system…………………………………………………...9 1.8 Cas9-mediated Base editors…………………………………………11 1.9 CRISPR/Cas9 system in inactivation of HBV genomes….13 1.10 The potential and challenge for curing CHB using the CRISPR/Cas9 system…...........................................................14 Specific Aims……………………………………………………………………...16 Material and Methods 2.1 Plasmids………………………………………………………………………..17 2.2 Cell lines and culture…………………………………………………….18 2.3 Transfection of cell lines……………………………………………….19 2.4 Lentiviral production and transduction…………………………19 2.5 Transduction with lentiviruses………………………………………20 2.6 Preparation and infection of HBV…………………………………21 2.7 Sanger and MiSeq sequencing of base edited genomic DNA and cccDNA……….................................................................22 2.8 Immunoblotting assay………………………………………………….23 2.9 ELISA of HBsAg……………………………………………………………23 2.10 Quantitative real-time PCR (qRT-PCR)……………………...24 2.11 HBV DNA extraction and Southern blotting……………….24 2.12 DNA-library preparation and MiSeq sequencing………..25 2.13 Off-target assessment………………………………………………26 2.14 Immunofluorescence Assay……………………………………...27 2.15 DNA bisulfited and pyrosequencing assay for DNA methylation analysis……......................................................28 2.16 Statistics……………………………………………………………………29 Results 3.1 Application of Cas9-BE for treatment of CHB 3.1.1 To design and screen HBV-specific gRNAs for inducing nonsense mutations by spCas9 base editors……………………………………………………….......…………...30 3.1.2 Introducing nonsense mutations into integrated HBV genomes by base editing suppresses the expression of HBsAg and polymerase………………………………..............................31 3.1.3 To induce dual suppression of HBsAg and polymerase by HBV base-editing specific gRNAs of HBV genome…………………………………................…………………..34 3.1.4 To validate the dual suppression phenomenon by specific point mutations of HBV genome……………………………………………………………………….35 3.1.5 Suppression of HBV protein expression through base editing HBV cccDNA by spCas9 base editor………………………………………………………......……………37 3.1.6 Using the intein-mediated split Cas9-BE system via rAAV to base-editing HBV genomes…………………………………………………………………....38 3.2 Application of Cas9-mediated DNA methylation for CHB treatment 3.2.1 Suppressing the expression of HBV genome by CRISPR/Cas9-mediated de novo methylation………………………………………………………………...39 Discussion 4.1 Summary of this study……………………………………………42 4.2 Approaches to improve the base editing in HBV genomes……………………..............................................…..42 4.3 The advantages of Cas9-BE for inactivation of HBV genome………………....................................................……46 4.4 Kill two birds with one stone: dual suppression of two genes by base editing in the overlapping regions of HBV genome…………………….......................……………………………47 4.5 Silencing the HBV promoters by Cas9-DNA methyltransferase or prime editing.............................................................49 4.6 Obstacles and advancement of CRISRP-Cas9 technologies………………….........................................…...50 4.7 Conclusion and perspectives………………………………...53 References……………………………………….………………………...54 Figures Figure 1. Schematic illustration of the HBV life cycle.…..62 Figure 2. Current and future HBV virological targets for treatment and cure of CHB.............................................................63 Figure 3. Cytosine base editing………………………………….64 Figure 4. Screening gRNAs for SpCas9-mediated base editing in HBV-HEK293T cells……………………………………….............65 Figure 5. Screening gRNAs for SpCas9 variants-mediated base editing in HBV-HEK293T cells…………………………………….68 Figure 6. Sanger sequencing of base-edited sites in the polymerase and surface genes of HBV genome…………69 Figure 7. The base editing of specific HBV loci to effect viral gene expression of HepG2.2.15 cells………………………....……..70 Figure 8. The off-target analysis for the top three genomic loci of two gRNAs gP9 and gS8 by BE4-mediated SpCas9 in HepG2.2.15 cells………………………………….........................................72 Figure 9. Dual suppression of HBsAg and polymerase by SpCas9 base editors……........................................................74 Figure 10. Validation of the effect of base-edited missense mutations on the expression of HBV surface and polymerase proteins in Huh7………………………………..................……76 Figure 11. Base editing of HBV cccDNA………............79 Figure 12. Efficiency of delivery of BE3 and HBV infection in HepG2-NTCP cells………………………………….......……...83 Figure 13. The intein-mediated split–Cas9……………...84 Figure 14. The methylation levels in HBV genome…..85 Tables Table1. The base-editing efficiency of SpCas9-BE with individual gRNAs……….…..........................................................88 Table 2. The conserveness of gRNAs indifferent HBV genotypes…………………...........................................89 Table 3. The base-editing efficiency of SpCas9-BE variant VQR, VRER, and EQR with individual gRNAs………………….90 Table 4. Plasmid list and function…………………………92 Table 5. Primer and probe list…………………………...…93 Appendix Figure S1. Mechanisms of Cas9-mediated prime editing…………………………….......................................97 | |
dc.language.iso | en | |
dc.title | 發展非切割式CRISPR/Cas9衍生之基因編輯系統治療慢性B型肝炎 | zh_TW |
dc.title | Development of the CRISPR/Cas9-mediated non-cleavage gene-editing strategies for treatment of chronic hepatitis B | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 凌嘉鴻,陳佑宗,蔡錦華,葉秀慧 | |
dc.subject.keyword | B型肝炎病毒,共價閉合環狀去氧核糖核酸,單鹼基編輯器,先導編輯,前間隔序列鄰近構形, | zh_TW |
dc.subject.keyword | hepatitis B virus,cccDNA,CRISPR/Cas9,base editors,split-Cas9, | en |
dc.relation.page | 97 | |
dc.identifier.doi | 10.6342/NTU202000737 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2020-04-10 | |
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 |
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