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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 | Michael Sheng-Fu Feng | en |
dc.contributor.author | 馮聖富 | zh_TW |
dc.date.accessioned | 2021-05-11T04:57:57Z | - |
dc.date.available | 2019-08-18 | |
dc.date.available | 2021-05-11T04:57:57Z | - |
dc.date.copyright | 2019-08-18 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-08 | |
dc.identifier.citation | McBride, H. M., Neuspiel, M. & Wasiak, S. Mitochondria: more than just a powerhouse. Curr. Biol. 16, R551–60 (2006).
2. Pittis, A. A. & Gabaldón, T. Late acquisition of mitochondria by a host with chimaeric prokaryotic ancestry. Nature 531, 101–104 (2016). 3. Gaziev, A. I. & Shaĭkhaev, G. O. [Nuclear mitochondrial pseudogenes]. Mol. Biol. (Mosk.) 44, 405–417 (2010). 4. Wallace, D. C. Mitochondrial genetic medicine. Nat. Genet. 50, 1642–1649 (2018). 5. Bacman, S. R., Williams, S. L., Duan, D. & Moraes, C. T. Manipulation of mtDNA heteroplasmy in all striated muscles of newborn mice by AAV9-mediated delivery of a mitochondria-targeted restriction endonuclease. Gene Ther 19, 1101–1106 (2011). 6. Bacman, S. R., Williams, S. L., Pinto, M., Peralta, S. & Moraes, C. T. Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs. Nature Medicine 2013 19:9 19, 1111–1113 (2013). 7. Gammage, P. A., Rorbach, J., Vincent, A. I., Rebar, E. J. & Minczuk, M. Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations. EMBO Mol Med 6, 458–466 (2014). 8. Man, P. Y. W., Turnbull, D. M. & Chinnery, P. F. Leber hereditary optic neuropathy. Journal of Medical Genetics 39, 162–169 (2002). 9. Rahman, S. et al. Leigh syndrome: Clinical features and biochemical and DNA abnormalities. Ann. Neurol. 39, 343–351 (1996). 10. Lake, N. J., Compton, A. G., Rahman, S. & Thorburn, D. R. Leigh syndrome: One disorder, more than 75 monogenic causes. Ann. Neurol. 79, 190–203 (2016). 11. Wallace, D. C. Why Do We Still Have a Maternally Inherited Mitochondrial DNA? Insights from Evolutionary Medicine. Annual Review of Biochemistry 76, 781–821 (2007). 12. Schwartz, M. & Vissing, J. New patterns of inheritance in mitochondrial disease. Biochem. Biophys. Res. Commun. 310, 247–251 (2003). 13. Tuppen, H. A. L., Blakely, E. L., Turnbull, D. M. & Taylor, R. W. Mitochondrial DNA mutations and human disease. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1797, 113–128 (2010). 14. Jenuth, J. P., Peterson, A. C. & Shoubridge, E. A. Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice. Nat. Genet. 16, 93–95 (1997). 15. Alexeyev, M. F. et al. Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes. Gene Ther 46, 428–8 (2008). 16. Bacman, S. R., Williams, S. L., Garcia, S. & Moraes, C. T. Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease. Gene Ther 17, 713–720 (2010). 17. Reddy, P. et al. Selective Elimination of Mitochondrial Mutations in the Germline by Genome Editing. Cell 161, 459–469 (2015). 18. Van Houten, B., Hunter, S. E. & Meyer, J. N. Mitochondrial DNA damage induced autophagy, cell death, and disease. Front Biosci (Landmark Ed) 21, 42–54 (2016). 19. Patananan, A. N., Wu, T.-H., Chiou, P.-Y. & Teitell, M. A. Modifying the Mitochondrial Genome. Cell Metabolism 23, 785–796 (2016). 20. Tanaka, M. et al. Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria. J. Biomed. Sci. 9, 534–541 (2002). 21. Bayona-Bafaluy, M. P., Blits, B., Battersby, B. J., Shoubridge, E. A. & Moraes, C. T. Rapid directional shift of mitochondrial DNA heteroplasmy in animal tissues by a mitochondrially targeted restriction endonuclease. Proc. Natl. Acad. Sci. U.S.A. 102, 14392–14397 (2005). 22. Smith, J. et al. Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res. 28, 3361–3369 (2000). 23. Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731–734 (2011). 24. Sung, Y. H. et al. Knockout mice created by TALEN-mediated gene targeting. Nat. Biotechnol. 31, 23–24 (2013). 25. Dutta, S., Madan, S. & Sundar, D. Exploiting the recognition code for elucidating the mechanism of zinc finger protein-DNA interactions. BMC Genomics 17, 1037 (2016). 26. Minczuk, M., Papworth, M. A., Miller, J. C., Murphy, M. P. & Klug, A. Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA. Nucleic Acids Res. 36, 3926–3938 (2008). 27. Hashimoto, M. et al. MitoTALEN: A General Approach to Reduce Mutant mtDNA Loads and Restore Oxidative Phosphorylation Function in Mitochondrial Diseases. Mol. Ther. 23, 1592–1599 (2015). 28. Wright, A. V., Nuñez, J. K. & Doudna, J. A. Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering. Cell 164, 29–44 (2016). 29. Jinek, M. et al. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337, 816–821 (2012). 30. Jo, A. et al. Research Article Efficient Mitochondrial Genome Editing by CRISPR/Cas9. BioMed Research International 1–10 (2015). doi:10.1155/2015/305716 31. Gammage, P. A., Moraes, C. T. & Minczuk, M. Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized. Trends Genet. (2017). doi:10.1016/j.tig.2017.11.001 32. Loutre, R., Heckel, A.-M., Smirnova, A., Entelis, N. & Tarassov, I. Can Mitochondrial DNA be CRISPRized: Pro and Contra. IUBMB Life 85, 133 (2018). 33. Bian, W.-P. et al. Knock-In Strategy for Editing Human and Zebrafish Mitochondrial DNA Using Mito-CRISPR/Cas9 System. ACS Synth Biol 8, 621–632 (2019). 34. Schumann, K. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc. Natl. Acad. Sci. U.S.A. 112, 10437–10442 (2015). 35. Zhou, J., Liu, L. & Chen, J. Mitochondrial DNA heteroplasmy in Candida glabrata after mitochondrial transformation. Eukaryotic Cell 9, 806–814 (2010). 36. Mileshina, D., Koulintchenko, M., Konstantinov, Y. & Dietrich, A. Transfection of plant mitochondria and in organello gene integration. Nucleic Acids Res. 39, e115–e115 (2011). 37. D'Aurelio, M. et al. Heterologous mitochondrial DNA recombination in human cells. Hum. Mol. Genet. 13, 3171–3179 (2004). 38. Gilkerson, R. W., Schon, E. A., Hernandez, E. & Davidson, M. M. Mitochondrial nucleoids maintain genetic autonomy but allow for functional complementation. J. Cell Biol. 181, 1117–1128 (2008). 39. Bacman, S. R., Williams, S. L. & Moraes, C. T. Intra- and inter-molecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks. Nucleic Acids Res. 37, 4218–4226 (2009). 40. Lakshmipathy, U. & Campbell, C. Double strand break rejoining by mammalian mitochondrial extracts. Nucleic Acids Res. 27, 1198–1204 (1999). 41. Srivastava, S. & Moraes, C. T. Double-strand breaks of mouse muscle mtDNA promote large deletions similar to multiple mtDNA deletions in humans. Hum. Mol. Genet. 14, 893–902 (2005). 42. Andreyev, A. Y., Kushnareva, Y. E. & Starkov, A. A. Mitochondrial metabolism of reactive oxygen species. Biochemistry Mosc. 70, 200–214 (2005). 43. Kukat, A. et al. Generation of rho0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses. Nucleic Acids Res. 36, e44 (2008). 44. Chacinska, A., Koehler, C. M., Milenkovic, D., Lithgow, T. & Pfanner, N. Importing mitochondrial proteins: machineries and mechanisms. Cell 138, 628–644 (2009). 45. Moberg, P. et al. NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia. J. Mol. Biol. 336, 1129–1140 (2004). 46. Saitoh, T. et al. Tom20 recognizes mitochondrial presequences through dynamic equilibrium among multiple bound states. EMBO J. 26, 4777–4787 (2007). 47. Yamano, K. et al. Tom20 and Tom22 share the common signal recognition pathway in mitochondrial protein import. J. Biol. Chem. 283, 3799–3807 (2008). 48. Tamura, Y. et al. Tim23-Tim50 pair coordinates functions of translocators and motor proteins in mitochondrial protein import. J. Cell Biol. 184, 129–141 (2009). 49. Jeandard, D. et al. Import of Non-Coding RNAs into Human Mitochondria: A Critical Review and Emerging Approaches. Cells 8, 286 (2019). 50. Rubio, M. A. T. et al. Mammalian mitochondria have the innate ability to import tRNAs by a mechanism distinct from protein import. Proc. Natl. Acad. Sci. U.S.A. 105, 9186–9191 (2008). 51. Mercer, T. R. et al. The human mitochondrial transcriptome. Cell 146, 645–658 (2011). 52. Wang, G. et al. PNPASE regulates RNA import into mitochondria. Cell 142, 456–467 (2010). 53. Cameron, T. A., Matz, L. M. & De Lay, N. R. Polynucleotide phosphorylase: Not merely an RNase but a pivotal post-transcriptional regulator. PLoS Genet. 14, e1007654 (2018). 54. Mohanty, B. K. & Kushner, S. R. Polynucleotide phosphorylase functions both as a 3‘ right-arrow 5’ exonuclease and a poly(A) polymerase in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 97, 11966–11971 (2000). 55. Yehudai-Resheff, S., Hirsh, M. & Schuster, G. Polynucleotide phosphorylase functions as both an exonuclease and a poly(A) polymerase in spinach chloroplasts. Mol. Cell. Biol. 21, 5408–5416 (2001). 56. Golzarroshan, B. et al. Crystal structure of dimeric human PNPase reveals why disease-linked mutants suffer from low RNA import and degradation activities. Nucleic Acids Res. 46, 8630–8640 (2018). 57. Fox, T. D. Mitochondrial protein synthesis, import, and assembly. Genetics 192, 1203–1234 (2012). 58. Becker, T., Böttinger, L. & Pfanner, N. Mitochondrial protein import: from transport pathways to an integrated network. Trends Biochem. Sci. 37, 85–91 (2012). 59. Wada, K.-I., Hosokawa, K., Ito, Y. & Maeda, M. Quantitative control of mitochondria transfer between live single cells using a microfluidic device. Biol Open 6, 1960–1965 (2017). 60. Su, X. & Dowhan, W. Translational regulation of nuclear gene COX4 expression by mitochondrial content of phosphatidylglycerol and cardiolipin in Saccharomyces cerevisiae. Mol. Cell. Biol. 26, 743–753 (2006). 61. Abu-Elheiga, L. et al. The subcellular localization of acetyl-CoA carboxylase 2. Proc. Natl. Acad. Sci. U.S.A. 97, 1444–1449 (2000). 62. Bravo-Alonso, I. et al. Nonketotic hyperglycinemia: Functional assessment of missense variants in GLDC to understand phenotypes of the disease. Hum. Mutat. 38, 678–691 (2017). 63. Bogenhagen, D. F., Rousseau, D. & Burke, S. The layered structure of human mitochondrial DNA nucleoids. J. Biol. Chem. 283, 3665–3675 (2008). 64. Prasannan, P., Pike, S., Peng, K., Shane, B. & Appling, D. R. Human mitochondrial C1-tetrahydrofolate synthase: gene structure, tissue distribution of the mRNA, and immunolocalization in Chinese hamster ovary calls. J. Biol. Chem. 278, 43178–43187 (2003). 65. Walkup, A. S. & Appling, D. R. Enzymatic characterization of human mitochondrial C1-tetrahydrofolate synthase. Arch. Biochem. Biophys. 442, 196–205 (2005). 66. Wang, G., Shimada, E., Koehler, C. M. & Teitell, M. A. PNPASE and RNA trafficking into mitochondria. Biochim. Biophys. Acta 1819, 998–1007 (2012). 67. Wang, G. et al. PNPASE Regulates RNA Import into Mitochondria. Cell 142, 456–467 (2010). 68. Smirnov, A., Entelis, N., Martin, R. P. & Tarassov, I. Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18. Genes Dev. 25, 1289–1305 (2011). 69. Kolesnikova, O. et al. Selection of RNA aptamers imported into yeast and human mitochondria. RNA 16, 926–941 (2010). 70. Gowher, A., Smirnov, A., Tarassov, I. & Entelis, N. Induced tRNA import into human mitochondria: implication of a host aminoacyl-tRNA-synthetase. PLoS ONE 8, e66228 (2013). 71. Wang, G. et al. Correcting human mitochondrial mutations with targeted RNA import. Proc. Natl. Acad. Sci. U.S.A. 109, 4840–4845 (2012). 72. Lee, N. S. et al. Functional and intracellular localization properties of U6 promoter-expressed siRNAs, shRNAs, and chimeric VA1 shRNAs in mammalian cells. RNA 14, 1823–1833 (2008). 73. Chen, B. et al. Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System. Cell 155, 1479–1491 (2013). 74. Kim, S. et al. CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 28, 367–373 (2018). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/handle/123456789/685 | - |
dc.description.abstract | 粒線體對於維持細胞內的能量及動態恆定扮演極重要的角色,當粒線體基因受損時會造成粒線體功能部分失活,甚至導致嚴重的遺傳性疾病,例如萊氏症(Leigh syndrome)。目前雖然有許多團隊嘗試利用限制酶或人工合成的DNA內切酶,例如:ZFN或TALEN,來針對突變的粒線體基因進行剪切剔除,但這些方法往往會受到選擇位點不足、製作不易等缺點所限制。近年來迅速發展的基因編輯技術CRISPR-Cas9便可解決上述兩項問題,目前除了三篇仍具有爭議的研究發表外,尚未有其他人提出相關研究成果或重複其研究。因此,在此篇研究中,我們分別在Cas9蛋白及嚮導RNA (guide RNA)上進行一系列不同粒線體標的序列(mitochondria-targeting sequence, MTS)的修飾。根據結果,我們發現帶有MTHFD1L MTS之mito-Cas9蛋白與僅帶有5’端10個核苷酸延長序列之嚮導RNA 51號,這兩者可以最有效率地進入粒線體。我們的結果可望運用CRISPR-Cas9技術來建立一套更加完善的粒線體基因編輯工具,未來將可應用於生物學研究與罕見粒線體疾病之治療。 | zh_TW |
dc.description.abstract | The mitochondrial genome is responsible for the maintenance of the cellular energy source and homeostasis. Therefore, partial loss of mitochondrial functionality and even devastating diseases happen when the mitochondrial genome is damaged. Nowadays, several strategies like restriction enzymes, ZFNs and TALENs, have been developed to specifically eliminate mutated mitochondrial DNA. However, all methods aforementioned have their own limitations on either limited target site choice or laborious manufactural process. CRISPR-Cas9 is a soaring new DNA editing tool which has not been widely applied in mitochondrial genome engineering, except for three controversial papers published. Consequently, in this study, we aim to establish a more reliable and tractable platform by fusing both Cas9 protein and guide RNA with various mitochondria-targeting sequences (MTSs). Our data show that the mito-Cas9 with the MTS from mitochondrial monofunctional C1-tetrahydrofolate synthase (MTHFD1L) and the mito-guide RNA 51 with only 10-nucleotide extended form the 5’ end both have the best import efficiency into mitochondria. Our study provides a potential technique to edit mitochondrial genome through CRISPR-Cas9 gene tool and may help generate gene therapy for mitochondrial diseases. | en |
dc.description.provenance | Made available in DSpace on 2021-05-11T04:57:57Z (GMT). No. of bitstreams: 1 ntu-108-R06b46008-1.pdf: 11321652 bytes, checksum: c5dbfc68b788d5ddd8008fa44c2b8ad0 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 謝辭 2
中文摘要 3 Abstract 4 Introduction 8 1. Significance of mitochondria 8 2. Diseases caused by pathogenic mtDNA mutations 9 3. Current status in mitochondrial genome manipulation 10 4. CRISPR-Cas9 gene editing technology and its application on mtDNA 11 5. DNA double-strand break repair mechanism in mammalian mitochondria 13 6. Mitochondrial protein import machinery 14 7. Mitochondrial RNA import machinery 15 8. Specific aim of this study 16 Results 17 1. Folded protein cannot be transported into mitochondria 17 2. A novel MTS is able to import mito-Cas9 into mitochondria 18 3. Putative RNA transporter and its counterpart RNAs exist in HeLa mitochondria 20 4. Structures from nucleus-encoded mitochondrial RNAs maintain Cas9 cleavage 21 5. Mito-sgRNA with only linker on 5’ end performs the best import 22 6. Cellular abnormality observed after sorting of mito-Cas9-GFP-positive cells 23 Discussion 25 1. Previous research on mtDNA editing by CRISPR-Cas9 system 25 2. Cas9 can only enter mitochondria with the assistance of a novel MTS 26 3. All modifications on guide RNA show no complete impediment on Cas9 cleavage ability 27 4. Guide RNA with only linker shows unexpectedly high mitochondrial import rate 28 5. Low expression level of mito-Cas9 may hinder downstream experiments 29 6. Recommendation for future research 29 Materials and Methods 31 1. Bacterial strains, primers and plasmids 31 2. Competent cell preparation 31 3. Plasmid construction 32 4. Cell culture 35 5. Nucleofection 36 6. Immunofluorescence 36 7. Synthesis of RNA by T7 in vitro transcription 37 8. Calf intestinal alkaline phosphatase (CIP) treatment 40 9. In vitro cleavage assay 41 10. RNA transfection 42 11. Dynabead mitochondrial extraction 43 12. MACS mitochondrial extraction 44 13. RNA extraction 46 14. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) 46 Reference 98 | |
dc.language.iso | en | |
dc.title | CRISPR-Cas9技術應用於粒線體基因編輯之優化設計 | zh_TW |
dc.title | Repurposing CRISPR-Cas9 Technology for Mitochondrial Genome Editing | en |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊維元(Wei-Yuan Yang),張壯榮(Chuang-Rung Chang) | |
dc.subject.keyword | 粒線體疾病,CRISPR-Cas9,基因治療,粒線體RNA,PNPase, | zh_TW |
dc.subject.keyword | Mitochondrial genome editing,mitochondria-targeting Cas9,mitocondria RNA import,PNPase, | en |
dc.relation.page | 101 | |
dc.identifier.doi | 10.6342/NTU201902787 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2019-08-08 | |
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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