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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 凌嘉鴻 | |
| dc.contributor.author | Hsin-Ju Hung | en |
| dc.contributor.author | 洪欣如 | zh_TW |
| dc.date.accessioned | 2021-06-17T08:27:25Z | - |
| dc.date.available | 2029-08-12 | |
| dc.date.copyright | 2019-08-19 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-12 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74274 | - |
| dc.description.abstract | 線粒體是真核細胞中的負責產生能量的胞器，由於產生能量的過程中會進行氧化反應使線粒體DNA（mtDNA）更容易受到損傷和突變。超過50種疾病是由mtDNA突變引起的，難以被研究且幾乎不可能治愈。目前已知可編程核酸酶如ZFN和TALEN已用於靶向mtDNA（主要用於消除突變mtDNA），但設計和構建的過程耗時，相比之下，由RNA引導的CRISPR基因技術更簡單。在本論文中，我的目的是設計CRISPR-Cpf1以用於人類mtDNA編輯。CRISPR-Cpf1系統比CRISPR-Cas9具有幾個優點，Cpf1具有較短的crRNA，可以更好地進入線粒體，此外，Cpf1具有RNA核酸酶活性，其可以將一系列crRNA加工成相同或多重靶序列的單個crRNA。最後，Cpf1切割產生交錯切割，以通過微同源重組介導的末端接合（MMEJ）使DNA有機會插入，據報導MMEJ是人線粒體中唯一的雙股斷裂（DSB）修復途徑。為了進入和編輯線粒體基質中的mtDNA，有必要設計靶向線粒體的Cpf1和crRNA（分別稱為mito-Cpf1和mito-crRNA）。為此，我篩選了幾種蛋白質和RNA的線粒體靶向序列，並通過提取線粒體、西方墨點法、免疫熒光和RT-qPCR驗證了mito-Cpf1和mito-crRNA的定位。下一步是在線粒體中表現Cpf1複合物以靶向和切割mtDNA上的特定位點。我的目標是檢測mtDNA切割的證據以及觀察切割後線粒體氧化呼吸的減少。開發CRISPR靶向mtDNA編輯系統將為mtDNA研究開闢許多可能性，並為mtDNA突變的治療提供更多機會。 | zh_TW |
| dc.description.abstract | Mitochondria are power generators in eukaryotic cells, but the oxidative reaction makes mitochondrial DNA (mtDNA) more susceptible to damages and leads to mutations. More than 50 diseases are caused by mtDNA mutations, and they are difficult to study and nearly impossible to cure. Programmable nucleases such as ZFN and TALEN have been used to target mtDNA (mostly for depletion of mutant mtDNA), but the design and construction are labor-intensive and time-consuming. By contrast, RNA-guided CRISPR gene technology is simpler. In this thesis, I aimed to engineer CRISPR-Cpf1 for human mtDNA editing. CRISPR-Cpf1 system has several advantages over CRISPR-Cas9. Cpf1 has shorter crRNA that may allow better entry into the mitochondria. In addition, Cpf1 has RNA nuclease activity that can process an array of crRNAs into individual crRNAs of the same or multiplex target sequences. Finally, Cpf1 cleavage generates stagger cuts to potentially allow DNA insertion via microhomology-mediated end joining (MMEJ), which is reported to be the only active double-strand break (DSB) repair pathway in human mitochondria. To reach and edit mtDNA in the mitochondrial matrix, it is necessary to engineer mitochondria-targeting Cpf1 and crRNA (termed mito-Cpf1 and mito-crRNA, respectively). To achieve this, I have screened several protein and RNA mitochondrial targeting sequences, and validated the localization of mito-Cpf1 and mito-crRNA by mitochondrial extraction, Western blotting, immunofluorescent microscopy and RT-qPCR. My next step was to reconstitute active Cpf1 complexes in mitochondria to target and cleavage specific sites on mtDNA. My goal was to detect evidence of mtDNA cleavage and subsequent reduction in mitochondrial oxidative respiration. A robust CRISPR-based mtDNA editing system would open many possibilities to study mtDNA maintenance and to pave the path for therapeutic editing of mtDNA mutations. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T08:27:25Z (GMT). No. of bitstreams: 1 ntu-108-R05b46012-1.pdf: 1929172 bytes, checksum: b7beb206b047261248248bf058c8d26d (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 中文摘要 i
英文摘要 ii 1. Introduction 1 1.1 Organization of mitochondria genome 1 1.2 Maintenance of mitochondrial DNA integrity 2 1.3 The current status of mitochondria editing 3 1.4 CRISPR-Cpf1 4 1.5 Mitochondria protein import 6 1.6 Mitochondria RNA import 8 1.7 Specific aim 9 2. Results 11 2.1 Mito-Cpf1 distribution to mitochondria 11 2.2 Optimized the isolation of mitochondria through sonication and magnetic beads 12 2.3 Mitochondria targeting sequence may affect Mito-crRNA folding 14 2.4 RNaseA/T1 effective eliminated interference RNA adhere to mitochondria outside 15 2.5 Mito-crRNA import to mitochondria 17 3. Discussion 18 4. Materials and Methods 21 4.1 Cell culture 21 4.2 Plasmid construction 21 4.3 Lentiviral production and concentration 22 4.4 Lentiviral transduction 23 4.5 Mitochondrial protein extraction 23 4.6 Western blot 24 4.7 Immunofluorescence Microscopy 25 4.8 In vitro synthesis of mito-crRNA arrays 25 4.9 In vitro cleavage assays 26 4.10 RNA transfection 26 4.11 RT-qPCR 27 5. Reference 28 6. Figures and Tables 31 圖目錄 Figure 1 31 Figure 2 32 Figure 3 33 Figure 4 35 Figure 5 36 Figure 6 37 Figure 7 38 Figure 8 39 Figure 9 40 Figure 10 41 Figure 11 42 表目錄 Table 1 43 Table 2 47 Table 3 50 Table 4 53 Table 5 54 Table 6 55 | |
| dc.language.iso | en | |
| dc.title | 建立編輯人體粒線體基因的CRISPR-Cpf1系統 | zh_TW |
| dc.title | Engineering of CRISPR-Cpf1 system for human mitochondrial genome editing | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊維元,張壯榮 | |
| dc.subject.keyword | 線粒體,線粒體DNA,CRISPR,Cpf1,線粒體基因組編輯, | zh_TW |
| dc.subject.keyword | mitochondria,mitochondrial DNA,CRISPR,Cpf1,mitochondrial genome editing, | en |
| dc.relation.page | 55 | |
| dc.identifier.doi | 10.6342/NTU201903090 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-08-13 | |
| 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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