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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 凌嘉鴻(Steven Lin) | |
dc.contributor.author | Max Chu | en |
dc.contributor.author | 朱銘 | zh_TW |
dc.date.accessioned | 2021-06-16T08:31:24Z | - |
dc.date.available | 2025-07-15 | |
dc.date.copyright | 2020-07-17 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-07-10 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58794 | - |
dc.description.abstract | 粒線體對於細胞進行能量代謝及維持動態恆定而言至關重要,當粒線體基因突變可能導致粒線體功能缺失,甚至引發嚴重疾病。粒線體基因病變與修復機制的研究具有高度挑戰性,而其仰賴於粒線體基因編輯工具的開發。過去有研究團隊嘗試利用限制酶、人工合成之DNA內切酶 (如:ZFN或TALENs)剔除突變之粒線體基因。近年來由於CRISPR-Cas9技術在目標基因位點設計及基因編輯之方便性等優勢下興起,此技術亦有開發於粒線體基因編輯之潛力,然而目前已知對於輸送嚮導RNA (guide RNA)至粒線體之細胞機制的研究並不深入。在這篇研究中,我們對Cas9蛋白以及嚮導RNA分別修飾粒線體標的序列(mitochondria targeting sequence; MTS),促使其有效率的進入粒線體中。我們在高解析度共軛焦顯微鏡下證實mito-Cas9能進入粒線體內。藉由反轉錄PCR分析,我們在粒線體萃取中發現有增量之mito-sgRNA。然而在開發應用於粒線體基因編輯之Cas9系統(mito-Cas9 system)時,我們發現在HeLa細胞中表現mito-Cas9會造成細胞毒性而導致細胞凋亡,其中存活的細胞則有關閉mito-Cas9表現之現象,且輸送mito-sgRNA至細胞中會造成粒線體基因數量減少。為了建立一套完善的基因編輯系統,我們致力對mito-Cas9的表達時間與強度進行優化以降低其造成的粒線體壓力。這套mito-Cas9系統的開發可望應用於粒線體之基因體研究以及粒線體基因病變之治療。 | zh_TW |
dc.description.abstract | Mitochondria are essential organelles in eukaryotic cells, indispensable for energy production, metabolisms and homeostasis. Mutations in mitochondrial genome (mtDNA) are highly disruptive to mitochondrial functions and cause devastating diseases. The research of mtDNA repair and mutation pathogenicity is challenging, and the lack of good genetic tools is one of the main reasons. Genetic editing of mtDNA is not a new idea and has been demonstrated by using restriction enzymes and programmable nucleases such as Zinc-finger nucleases, Transcription activator-like effector nucleases and more recently CRISPR-Cas9. Each technique has its strengths and limitations, and this work focuses on CRISPR-Cas9 system for mtDNA editing. RNA-guided DNA cleavage of Cas9 allows fast reprogramming of Cas9 target specificity. However, mitochondrial localization of the guide RNA requires specialized RNA import mechanisms that remain poorly understood. Here we describe the modifications of Cas9 protein (mito-Cas9) and guide RNA (mito-sgRNA) to achieve robust and specific mitochondrial localization. We present high-resolution confocal microscopic images of mitochondrial localization of mito-Cas9, which carries a novel mitochondrial targeting sequence. We also detected enrichment of mito-sgRNA, which carries a unique RNA hairpin, in the purified mitochondrial extract by reverse transcription-PCR using endogenous 5S rRNA as a benchmark. However, the reconstitution of mtDNA-targeting Cas9: guide RNA ribonucleoprotein complexes is difficult, owing to high cellular toxicity of mito-Cas9. The expression of mito-Cas9 alone in HeLa cell line by plasmid transfection induced mitochondrial fragmentation and cell death within two days. The surviving cells had completely silenced the expression of mito-Cas9 after three weeks. The transfection of in vitro transcribed mito-sgRNA caused reduced copies of mtDNA in HeLa cell line within one day. Strategies are needed to fine tune the expression level and timing of the mito-Cas9 system to avoid undesirable mitochondrial stress and enable unbiased detection of mtDNA cleavage. Successful establishment of a robust CRISPR-based mtDNA editing system will help advance mtDNA research and offer strategies for therapeutic correction of pathogenic mtDNA mutations. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T08:31:24Z (GMT). No. of bitstreams: 1 U0001-0907202017490900.pdf: 5612418 bytes, checksum: 9c6dddf95e3787e93bf1c476b5cb2521 (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | Table of Contents 口試委員會審定書 i 謝辭 ii 中文摘要 iv Abstract v Introduction 1 1. Significance of mitochondria 1 2. Gene editing strategies for mtDNA manipulation 1 3. CRISPR-Cas9 gene-editing technology 3 4. CRISPR-Cas9-based mtDNA editing 4 5. Mitochondrial protein import mechanism 5 6. Mitochondrial RNA import 6 7. Specific aims of this study 7 Results 8 1. A novel MTS for mito-Cas9 import into mitochondria 8 2. Mito-Cas9 expression leads to cellular abnormality 9 3. Mito-Cas9 transfection efficiency optimization 10 4. Doxycycline-inducible mito-Cas9 stable cell line construction 11 5. mito-Cas9 cell line preparation via puromycin selection 12 6. Modified mito-sgRNAs maintain Cas9 cleavage activity 13 7. PNPase and PNPase-mediated RNAs exist in HeLa mitochondria 14 8. Modified mito-sgRNAs facilitate mitochondria translocation 15 9. Mito-sgRNA transfection efficiency optimization 16 10. mtDNA targeting by mito-Cas9 system 17 11. Mitochondria physiology and cell viability influenced by mito-Cas9 system 17 Discussion 20 1. Previous studies on mtDNA editing by CRISPR-Cas9 system 20 2. A novel MTS facilitates mito-Cas9 translocation into mitochondria 21 3. Efficient mito-Cas9 gene expression requires optimized approaches 21 4. Mito-sgRNA with linker modification showed enhanced mitochondrial import efficiency 23 5. Mito-sgRNA interferes with mtDNA level within mitochondria 23 6. Perspectives on mtDNA targeting by mito-Cas9 system 24 Materials and Methods 25 1. Cell culture 25 2. Plasmid construction 25 3. Lipofection 26 4. Nucleofection 27 5. Immunofluorescence 27 6. Flow cytometry 28 7. Stable cell line construction via lentiviral transduction 28 8. Stable cell line construction via plasmid transfection 30 9. In situ RNA detection 30 10. RNA synthesis by T7 in vitro transcription 32 11. Calf intestinal alkaline phosphatase (CIP) treatment 34 12. RNA transfection 35 13. DNA extraction 36 14. Quantitative real-time polymerase chain reaction (qPCR) 36 15. WST-1 assay 36 16. Mito Stress Test 37 17. Statistical analysis 37 Reference 75 Appendix 78 Supplementary figure 1. Immunofluorescence images of mito-Cas9 78 Supplementary figure 2. Mito-Cas9 over-expression results in aberrant morphology 79 Supplementary figure 3. In vitro cleavage assay of mito-sgRNAs 80 Supplementary figure 4. PNPase exists in HeLa mitochondria 81 Supplementary figure 5. Schematic flow chart for mitochondrial RNA extraction 82 Supplementary figure 6. Mitochondrial translocation rate formulation 83 Supplementary figure 7. PNPase-mediated nuclear-encoded RNAs exist in HeLa mitochondria 84 Supplementary figure 8. Schematic flow chart for time-course abundance of mito-sgRNA51 in HeLa cells 85 Supplementary figure 9. Mito-sgRNA import efficiency 86 | |
dc.language.iso | en | |
dc.title | CRISPR-Cas9 技術於粒線體基因編輯之應用與對細胞功能之影響 | zh_TW |
dc.title | Opportunities and Challenges in Repurposing CRISPR- Cas9 System for Mitochondrial Genome Editing | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊維元(Wei Yuan Yang),陳光超(Guang-Chao Chen) | |
dc.subject.keyword | 粒線體疾病,基因編輯,CRISPR-Cas9,粒線體RNA,基因治療, | zh_TW |
dc.subject.keyword | Mitochondrial genome editing,CRISPR-Cas9,mitochondria-targeting Cas9,mitochondria RNA import,mito-Cas9 system, | en |
dc.relation.page | 86 | |
dc.identifier.doi | 10.6342/NTU202001420 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2020-07-13 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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