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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 凌嘉鴻 | |
| dc.contributor.author | An Yu | en |
| dc.contributor.author | 俞安 | zh_TW |
| dc.date.accessioned | 2021-06-17T08:29:37Z | - |
| dc.date.available | 2019-08-19 | |
| 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/123456789/74319 | - |
| dc.description.abstract | CRISPR-Cas9基因編輯技術目前應用相當廣泛，包括動植物以及微生物之領域。當 Cas9與短片段 DNA一同送入細胞之後， Cas9能切斷基因標靶位點，而DNA可作為同源重組修復之模板，以達到精準的基因編輯。先前研究發現：利用電穿孔技術將與人類基因組不同源的短片段 DNA與 Cas9送入細胞，在許多基因標靶位點上皆能顯著地提升基因編輯效率，而目前仍不明瞭造成此現象之原因。在我們的實驗中發現：短片段 DNA透過電穿孔的方式可以迅速地被送入細胞核之中，引起細胞內的 DNA損傷反應進而造成大規模地核小體重排。 Cas9的基因標靶位點被核小體佔據已經被證明是限制 Cas9作用的主要原因。由於短片段DNA刺激 DNA損傷反應而造成核小體重排，使得原本被核小體纏繞緊密的染色
質變得較為鬆散，故 Cas9較容易辨認並切斷標靶位點，進而增加基因編輯的效率。此研究說明了短片段 DNA提升基因編輯效率之機 制。探討進行基因編輯時， CRISPR-Cas9系統所引發的細胞反應，有助於開發更安全的基因編輯技術並同時兼具良好的編輯效率。 | zh_TW |
| dc.description.abstract | CRISPR-Cas9 is a robust genome editing technology that works in a broad range of organisms including human cells. When co-introduced with a synthetic DNA oligonucleotide template, Cas9 can facilitate site-specific integration of the DNA template sequence via homology-directed repair (HDR) for precise genome modification. An interesting observation was made in a previous study, in which the Cas9 gene editing efficiency in cells was significantly enhanced by co-electroporation of single-stranded DNA oligonucleotides. This enhancement was independent of DNA sequences. We discovered by serendipity that electroporation rapidly delivered DNA oligonucleotides into nucleus, triggered DNA damage response (DDR) and induced global nucleosome rearrangement. Since nucleosome occupancy is known to be the major impediment to target recognition by Cas9, decondensation of heterochromatin effectively lower this barrier by increasing the accessibility of DNA to Cas9, promoting double-strand break (DSB) repair, and resulting in higher Cas9 editing efficiency. To the best of our knowledge, this is the first report of nucleosome rearrangement by DNA electroporation, a common practice for general DNA delivery and nuclease-directed genome editing. A thorough investigation is therefore needed to evaluate the cellular consequences and safety and to determine the influence on genome editing outcome. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T08:29:37Z (GMT). No. of bitstreams: 1 ntu-108-R06b46006-1.pdf: 3137049 bytes, checksum: 288fc4a57ce70366548e15fa3e9fea4a (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | TABLE OF CONTENTS I
LIST OF TABLES IV 摘要 V ABSTRACT VI 1. INTRODUCTION 1 1.1 The mechanism of CRISPR-Cas9 1 1.2 Nucleosome impediment to Cas9 gene editing 2 1.3 Cracking the mystery of enhancement of Cas9 editing by DNA oligonucleotides 3 1.4 Introduction to DNA damage response 4 1.5 Research hypothesis and specific aims 6 2. RESULTS 7 2.1 γ-H2AX is induced by DNA oligonucleotides 7 2.2 DNA oligonucleotides enhance Cas9 editing efficiency 7 2.3 Aim 1 To define the location of DNA oligonucleotides 8 2.4 Aim 2 To ensure DNA oligonucleotides triggered DNA damage response 10 2.5 Aim 3 To proof nucleosome rearrangement induced by DNA oligonucleotides 12 3. DISCUSSION 13 4. MATERIALS AND METHODS 18 4.1 Cell lines and cell culture 18 4.2 Compound treatments 18 4.3 Preparation of sgRNA 19 4.4 Preparation of DNA oligonucleotides 21 4.5 Electroporation 22 4.5.1 X unit 22 4.5.2 Y unit 22 4.6 The cleavage of Cas9 target region analysis 23 4.6.1 In vitro cleavage assay 23 4.6.2 T7 endonuclease 1 Assay 23 4.7 Immunofluorescence staining and confocal microscopy 24 4.8 Western Blot Analysis 25 4.9 Micrococcal nuclease digestion 26 4.10 Synchronization and cell cycle analysis 27 4.11 RNA extraction and Real-time RT-PCR 28 5. APPENDIX 32 6. REFERENCE 34 7. FIGURES 41 Figure 1. Schematic representation of CRISPR-mediated genome editing 41 Figure 2. Schematic representation of DNA damage response 42 Figure 3. Schematic illustration of our hypothesis 43 Figure 4. DNA oligonucleotide-induced cellular response was similar with etoposide treatment 44 Figure 5. The impact of DNA oligonucleotides on Cas9 activity 45 Figure 6. The Cas9 editing efficiency was enhanced by DNA oligonucleotides 46 Figure 7. Experimental design of transfected DNA into cells in adherence by electroporation 47 Figure 8. The transfection efficiency and cell viability of electroporation on Lonza nucleofector Y unit 48 Figure 9. DNA damage response as induced by etoposide treatment 49 Figure 10. DNA oligonucleotides dispersed in nucleus 51 Figure 11. Kinetics of the DNA oligonucleotide-induced effects on DDR proteins 53 Figure 12. Effect of DNA oligonucleotides on HCT116 cell cycle distribution 54 Figure 13. Analysis of chromatin compaction by MNase digestion 55 Figure 14. CIP treatment eliminated IVT sgRNA-induced immune response 56 | |
| dc.language.iso | en | |
| dc.subject | CRISPR-Cas9 | zh_TW |
| dc.subject | 短片段 DNA | zh_TW |
| dc.subject | DNA損傷反應 | zh_TW |
| dc.subject | 核小體重排 | zh_TW |
| dc.subject | 基因編輯 | zh_TW |
| dc.subject | DNA damage response | en |
| dc.subject | DNA oligonucleotides | en |
| dc.subject | CRISPR-Cas9 | en |
| dc.subject | gene editing | en |
| dc.subject | nucleosome rearrangement | en |
| dc.title | 外源DNA造成的核小體短暫重排提升CRISPR-Cas9系統的基因編輯效率 | zh_TW |
| dc.title | Enhanced CRISPR editing by DNA oligonucleotide-induced transient nucleosome rearrangement | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳瑞華,冀宏源,張?仁 | |
| dc.subject.keyword | CRISPR-Cas9,短片段 DNA,DNA損傷反應,核小體重排,基因編輯, | zh_TW |
| dc.subject.keyword | CRISPR-Cas9,DNA oligonucleotides,DNA damage response,nucleosome rearrangement,gene editing, | en |
| dc.relation.page | 56 | |
| dc.identifier.doi | 10.6342/NTU201903055 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-08-12 | |
| 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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