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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳亘承 | zh_TW |
| dc.contributor.advisor | Hsuan-Chen Wu | en |
| dc.contributor.author | 黃章衍 | zh_TW |
| dc.contributor.author | Chang-Yen Huang | en |
| dc.date.accessioned | 2023-08-16T16:36:57Z | - |
| dc.date.available | 2023-11-09 | - |
| dc.date.copyright | 2023-08-16 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-09 | - |
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Roossinck, Editors. 2019, Academic Press. p. 33-70. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88977 | - |
| dc.description.abstract | 基因編輯系統CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins)自其發現以來，在醫療領域上具有重大應用與發展潛力，特別是在基因治療。然而CRISPR-Cas需要一個安全可靠、可廣泛、製造經濟化的運送系統，方以有效用於生物體的基因編輯與治療，目前的運送載體，例如病毒載體或人造聚合物往往面臨諸多挑戰，如安全因素或合成上的繁瑣複雜等。除此之外，CRISPR-Cas本身的遞送形式也是一個考量重點，爲了避免外源基因插入宿主基因體的風險，使用信使核糖核酸 (mRNA) 或核糖蛋白 (ribonucleoprotein, RNP) 是較爲安全的形式。本研究意在建立一種新型的益生菌之CRISPR-Cas RNP遞送系統，以益生菌衍生之奈米載體作爲包裹和傳遞RNP的載體。具體來說，益生菌Escherichia coli Nissle 1917 (EcN) 以基因敲落的方式，令其在分裂時產生不再分裂的微小細胞 (minicells)，同時讓EcN表達外源蛋白Cas12b使其在胞內組裝Cas12b RNP。這些微小細胞會藉由表達外膜蛋白invasin以結合哺乳類細胞表面，並藉由表現listeriolysin O來釋放細菌表達的蛋白進入細胞內部，一旦微小細胞被吞入並在細胞的胞內體中裂解，Cas12b RNP便可藉由核定位序列 (nucleus localization signal, NLS) 的幫助，運送到細胞核內完成基因編輯。同時在研究中，我們建構了CRISPR編輯報道細胞株 DGG.HeLa，以功能增益 (grain-of-function) 的方式來確認細胞是否有被成功編輯，在基因被成功編輯後，細胞才會生產綠色螢光蛋白 (green fluorescence protein; GFP) 。更具結果顯示，經由 minicells 運輸 Cas12b-sgRNA，我們能成功編輯 DGG.HeLa，並產生最高約5% 之永久綠色系細胞株。在未來我們將繼續調整 minicells 系統，增加編輯效率與生醫應用性。 | zh_TW |
| dc.description.abstract | CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) systems have an emerging utilization and research since its discovery, especially in gene therapy. A safe, universal, and affordable delivery platform is required when encountering in vivo editing. Current platforms such as viral vector and artificial polymer face several challenges, such as safety issues and complication in production. In addition, the form of delivered CRISPR-Cas is an issue to concern. It is commonly accepted that delivering RNP (ribonucleoprotein) and mRNA of CRIPSR-Cas are safer than delivering CRISPR-Cas DNA, to avoid possible gene integration. Here, we utilize a bioengineered probiotic-derived nanocarrier to deliver functional CRISPR-Cas12b RNP. Specifically, Escherichia coli Nissle 1917 (EcN) is engineered to produce non-proliferating minicells by gene knockout, as well as to express functional Cas12b RNP within the minicells. These minicells are capable of binding mammalian cells by expressing Yersinia outer membrane protein invasin, and are able to escape from endosome by expressing listeriolysin O. Once the minicells are engulfed and lysed within the targeting cell, Cas12b RNP would be transferred into nucleus by the assist of nuclear localization protein (NLS) to conduct further genome editing. In this study, we constructed a CRISPR-reporting cell line DGG.HeLa to specifically producing green fluorescence protein (GFP) only after CRISPR-editing via Cas12b-sgRNA minicells. As the result demonstrated in the research, the GFP-expressing stable DGG.HeLa cell line was created at the success rate of 5% via the engineered minicell system. We anticipate that further development of the CRISPR-minicell delivery system will increase the editing efficiency of future biomedical applications. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-08-16T16:36:57Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-08-16T16:36:57Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | Table of Content
Table of Content i Table of Figures iv 摘要 vii Abstract viii 1. Introduction 1 1.1 Gene Editing and CRISPR 2 1.1.1 CRISPR-Cas9 and CRISPR-Cas12b 3 1.2 CRISPR-Cas and Gene Therapy 5 1.2.1 CRISPR-Cas Delivery 5 1.3 Bacterial Vectors in Cell Delivery Platform 7 1.3.1 Probiotic E. coli Nissle 1917 (EcN) and delivery systems 9 1.3.2 Minicells as Delivery Platform 10 1.4 Specific Aims 11 2. Materials and Methods 13 2.1 Plasmid, Bacteria Strain and Mammalian Cell Line 13 2.1.1. Bacterial Strains 13 2.1.2 Cell Lines 14 2.1.3 Plasmids and Primers 14 2.2 Culture Medium and Other Reagents 17 2.2.1 Bacterial Medium and Drugs 17 2.2.2 Cell Culturing Medium and Drugs 19 2.3 Plasmid Construction and Transformation 20 2.3.1 pACYC-T5-mCherry (pT5RF) 20 2.3.2 pLAS3w.DsRed-eGFP’-eGFP’’.WPRE (DGG(WPRE)) 20 2.3.3 Preparation of Electrocompetent Cell and Electroporation 21 2.3.4 Development of DGG.HeLa Stable Cell Line 22 2.4 Production and Purification of Cas12b RNP in Minicell 23 2.4.1 Purification of Cas12b RNP 23 2.4.2 SDS-PAGE 25 2.4.3 In vitro DNA Cleavage Test 25 2.5 Production and Purification of Minicell 26 2.5.1 Determination of Contamination from Parental Cell 27 2.5.2 Growth Curve of EcN mini and EcN WT 28 2.5.3 Microscopy 28 2.6 Cell Culture and Minicell Co-Incubation Test 28 2.6.1 Cell Viability Test 28 2.6.2 Minicell Delivery Test 30 2.6.3 Lysozyme Treatment 30 2.6.4 Lysozyme and Polymyxin B Treatment 31 2.6.5 Immunofluorescence Staining 32 2.6.6. Transfection of Cas12b RNP by Lipofection 33 2.6.7 Transfection of Cas12b RNP by Electroporation 33 3. Results 35 3.1 Minicell Construction and Protein Production 35 3.1.1 Clarification of Minicell-Producing Strain EcN mini 35 3.1.2 Purification of Minicells 37 3.1.3 Production of Cas12b RNP in Minicell 41 3.2 Minicells on Cell Culture 45 3.2.1 Cytotoxicity of Minicells 45 3.2.2 Attachment of Minicell on Mammalian Cell by Invasin 49 3.2.3 Engulfment of Minicells by Invasin 49 3.2.4 Tracking Minicell Localization within HeLa Cells 54 3.2.5 Lysozyme and Polymyxin B Increased Minicell Binding 58 3.2.6 Cargo releasing and migration from Lysozyme and Polymyxin B pre-treated minicells after cellular uptake 62 3.3 Gene Editing 65 3.3.1 Establishment of DGG.HeLa Cell 65 3.3.2 Cas12b RNP Cleavage in Electroporated DGG.HeLa Cell 69 3.3.3 Cas12b RNP Delivery by Lysozyme and Polymyxin B-Treated Minicell 71 4. Conclusion and Discussion 74 5. Future Work and Perspective 80 6. Reference 82 7. Appendix 91 Table of Figures Figure 1. EcN mini and the produced minicells. 37 Figure 2. Establishment of minicell separation protocols. 40 Figure 3. Cas12b RNP purification and in vitro DNA cleavage activity test. 43 Figure 4. Cell viability test upon minicell coculture. 47 Figure 5. Binding and engulfment of inv-hly-expressing minicells by HeLa cells. 52 Figure 6. Minicell at different B/C ratio. 56 Figure 7. Minicell localization within HeLa cells. 57 Figure 8. The effect of lysozyme and polymyxin B pretreatment on the cellular binding of minicells. 61 Figure 9. The distribution of mCherry signal (from pre-treated minicells) after cellular uptake within 28 hours. 64 Figure 10. Establishment of DGG.HeLa cell line. 68 Figure 11. Cas12b RNP delivery for DGG.HeLa editing by electroporation and lipofection. 71 Figure 12. Cas12b RNP Delivery and DGG.HeLa cell editing by pretreated minicells. 73 Figure 13. pACYC2-T5lacO-mCherry 93 Figure 14. pACYC2-T5lacO-Cas12b-3NLS-sgEGFP 94 Figure 15. pGB2-inv-hly 95 Figure 16. pLAS3w-PNEO(DGG) 96 Figure 17. Merged image of transfected A549, 2 days after the transfection was conducted. 98 Figure 18. HeLa cells co-incubated with EcN mini 12Sp/GB2 at 37°C for 2 hours. 99 Figure 19. HeLa cells co-incubated with EcN mini 12Sp/GB2 at 4°C for 2 hours. 100 Figure 20. Minicell attachment at B/C 100 and 2000. 101 Figure 21. 144 hours post incubation.. 102 Table 1. Plasmids used in this research, its abbreviation, and its description. 15 Table 2. Colony forming unit after each purification step. 39 Table 3. Primers used in this research. 91 Table 4. G418 sensitivity of HEK293, A549, and HeLa cells 97 | - |
| dc.language.iso | en | - |
| dc.title | 以益生大腸桿菌衍生之奈米載體作爲CRISPR-Cas12b之遞送系統 | zh_TW |
| dc.title | Bactofection by Probiotic E. coli -Derived Nanocarriers for CRISPR-Cas12b Delivery | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 史有伶;黃楓婷 | zh_TW |
| dc.contributor.oralexamcommittee | Yu-Ling Shih;Feng-Ting Huang | en |
| dc.subject.keyword | CRISPR-Cas 遞送,基因編輯,E. coli Nissle 1917,微小細胞, | zh_TW |
| dc.subject.keyword | CRISPR-Cas delivery,gene editing,E. coli Nissle 1917,minciell, | en |
| dc.relation.page | 102 | - |
| dc.identifier.doi | 10.6342/NTU202303502 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2023-08-10 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科技學系 | - |
| dc.date.embargo-lift | 2024-08-01 | - |
| 顯示於系所單位： | 生化科技學系 | |
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