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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨(Ching-Tsan Huang) | |
| dc.contributor.author | Meng-Hsi Tsai | en |
| dc.contributor.author | 蔡孟羲 | zh_TW |
| dc.date.accessioned | 2021-05-13T06:39:58Z | - |
| dc.date.available | 2019-08-14 | |
| dc.date.available | 2021-05-13T06:39:58Z | - |
| dc.date.copyright | 2017-08-14 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-01 | |
| dc.identifier.citation | 1. Kim, Y. G., Cha, J., & Chandrasegaran, S. (1996). Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain. Proceedings of the National Academy of Sciences, 93(3), 1156-1160.
2. Cradick, T. J., Ambrosini, G., Iseli, C., Bucher, P., & McCaffrey, A. P. (2011). ZFN-site searches genomes for zinc finger nuclease target sites and off-target sites. BMC bioinformatics, 12(1), 152. 3. Hopkins, C. M., White, F. F., Choi, S. H., Guo, A., & Leach, J. E. (1992). Identification of a family of avirulence genes from Xanthomonas oryzae pv. oryzae. Molecular Plant-Microbe Interactions, 5(6), 451-459. 4. Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye, T., Nickstadt, A., & Bonas, U. (2009). Breaking the code of DNA binding specificity of TAL-type III effectors. Science, 326(5959), 1509-1512. 5. Liu, B., Zhang, A. K., Zhou, L., & Miao, J. (2015). New era of gene editing: a brief discussion of engineered nucleases with gene editing ability. Gene and Gene Editing, 1(1), 26-30. 6. Rudin, N., Sugarman, E., & Haber, J. E. (1989). Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics, 122(3), 519-534. 7. Mout, R., Ray, M., Lee, Y. W., Scaletti, F., & Rotello, V. M. (2017). In vivo delivery of CRISPR/Cas9 for therapeutic gene editing: progress and challenges. Bioconjugate Chemistry. 8. Waltz, E. (2016). CRISPR-edited crops free to enter market, skip regulation. 9. Wiedenheft, B., Sternberg, S. H., & Doudna, J. A. (2012). RNA-guided genetic silencing systems in bacteria and archaea. Nature, 482(7385), 331-338. 10. Wright, A. V., Nuñez, J. K., & Doudna, J. A. (2016). Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell, 164(1), 29-44. 11. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816-821. 12. Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., & Doudna, J. A. (2014). DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 507(7490), 62-67. 13. Pyne, M. E., Bruder, M. R., Moo-Young, M., Chung, D. A., & Chou, C. P. (2016). Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium. Scientific reports, 6. 14. Gophna, U., Allers, T., & Marchfelder, A. (2017). Finally, archaea get their CRISPR-Cas toolbox. Trends in Microbiology. 25(6), 430-432. 15. Qin, H., Xiao, H., Zou, G., Zhou, Z., & Zhong, J. J. (2017). CRISPR-Cas9 assisted gene disruption in the higher fungus Ganoderma species. Process Biochemistry. 56, 57-61. 16. Belhaj, K., Chaparro-Garcia, A., Kamoun, S., & Nekrasov, V. (2013). Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant methods, 9(1), 39. 17. Liang, P., Xu, Y., Zhang, X., Ding, C., Huang, R., Zhang, Z., Lv, J., Xie, X., Chen, Y., Li, Y., Bai, Y., Songyang, Z., Ma, W., Zhou, C., Huang, J., & Sun, Y. (2015). CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein & cell, 6(5), 363-372. 18. Shen, B., Zhang, W., Zhang, J., Zhou, J., Wang, J., Chen, L., Wang, L., Hodgkins, A., lyer, V., Huang, X., & Skarnes, W. C. (2014). Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nature methods, 11(4), 399-402. 19. Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173-1183. 20. Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., & Lim, W. A. (2013). CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 154(2), 442-451. 21. McDonald, J. I., Celik, H., Rois, L. E., Fishberger, G., Fowler, T., Rees, R., Kramer, A., Martens, A., Edwards, J. R., & Challen, G. A. (2016). Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biology open, 5, 866–874. 22. Mikuni, T., Nishiyama, J., Sun, Y., Kamasawa, N., & Yasuda, R. (2016). High-throughput, high-resolution mapping of protein localization in mammalian brain by in vivo genome editing. Cell, 165(7), 1803-1817. 23. Zhang, J. P., Li, X. L., Neises, A., Chen, W., Hu, L. P., Ji, G. Z., Yu, J. Y., Xu, J., Yuan, W. P., Cheng, T., & Zhang, X. B. (2016). Different effects of sgRNA length on CRISPR-mediated gene knockout efficiency. Scientific reports, 6. 24. Tycko, J., Myer, V. E., & Hsu, P. D. (2016). Methods for optimizing CRISPR-Cas9 genome editing specificity. Molecular cell, 63(3), 355-370. 25. Kleinstiver, B. P., Pattanayak, V., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Zheng, Z., & Joung, J. K. (2016). High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature, 529(7587), 490-495. 26. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., & Joung, J. K. (2014). Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nature biotechnology, 32(3), 279-284. 27. Baumann, M. (2016). CRISPR/Cas9 genome editing–new and old ethical issues arising from a revolutionary technology. NanoEthics, 10(2), 139-159. 28. Ahmad, M., Hirz, M., Pichler, H., & Schwab, H. (2014). Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Applied Microbiology and Biotechnology, 98(12), 5301–5317. 29. Itakura, K., Hirose, T., Crea, R., Riggs, A. D., Heyneker, H. L., Bolivar, F., & Boyer, H. W. (1977). Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science, 198(4321), 1056-1063. 30. Baroni, C. D., De Franceschi, G. S., Uccini, S., Adorini, L., Cnen, G. D., & Ruco, L. (1976). Biological effects of Escherichia coli lipopolysaccharide (LPS) in vivo. I. Selection in the mouse thymus of killer and helper cells. Immunology, 31(2), 217. 31. Bandaranayake, A. D., & Almo, S. C. (2014). Recent advances in mammalian protein production. FEBS letters, 588(2), 253-260. 32. Hartner, F. S., & Glieder, A. (2006). Regulation of methanol utilisation pathway genes in yeasts. Microbial Cell Factories, 5(1), 39. 33. Liang, S., Wang, B., Pan, L., Ye, Y., He, M., Han, S., Zheng, S., Wang, X., & Lin, Y. (2012). Comprehensive structural annotation of Pichia pastoris transcriptome and the response to various carbon sources using deep paired-end RNA sequencing. BMC genomics, 13(1), 738. 34. Cregg, J. M., Vedvick, T. S., & Raschke, W. C. (1993). Recent advances in the expression of foreign genes in Pichia pastoris. Nature Biotechnology, 11(8), 905-910. 35. Wang, T. Y., Huang, C. J., Chen, H. L., Ho, P. C., Ke, H. M., Cho, H. Y., Ruan, S. K., Hung, K. Y., Wang, I. L., Cai, Y. W., & Sung, H. M. (2013). Systematic screening of glycosylation-and trafficking-associated gene knockouts in Saccharomyces cerevisiae identifies mutants with improved heterologous exocellulase activity and host secretion. BMC Biotechnology, 13(1), 71. 36. Vassileva, A., Chugh, D. A., Swaminathan, S., & Khanna, N. (2001). Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter. Journal of biotechnology, 88(1), 21-35. 37. Waterham, H. R., Digan, M. E., Koutz, P. J., Lair, S. V., & Cregg, J. M. (1997). Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene, 186(1), 37-44. 38. Zhang, A. L., Luo, J. X., Zhang, T. Y., Pan, Y. W., Tan, Y. H., Fu, C. Y., & Tu, F. Z. (2009). Recent advances on the GAP promoter derived expression system of Pichia pastoris. Molecular biology reports, 36(6), 1611-1619. 39. Crook, N. C., Schmitz, A. C., & Alper, H. S. (2013). Optimization of a yeast RNA interference system for controlling gene expression and enabling rapid metabolic engineering. ACS synthetic biology, 3(5), 307-313. 40. Lee, Y., Kim, M., Han, J., Yeom, K.-H., Lee, S., Baek, S. H., & Kim, V. N. (2004). MicroRNA genes are transcribed by RNA polymerase II. The EMBO Journal, 23(20), 4051–4060. 41. Willis, I. M. (1993). RNA polymerase III. Genes, factors and transcriptional specificity. European Journal of Biochemistry, 212, 1–11. 42. Herr, A.J., Jensen, M.B., Dalmay, T., Baulcombe, D.C. (2005). RNA polymerase IV directs silencing of endogenous DNA. Science 308: 118–120. 43. Wierzbicki, A. T., Ream, T., Haag, J. R., & Pikaard, C. S. (2009). RNA Polymerase V transcription guides ARGONAUTE4 to chromatin. Nature Genetics, 41(5), 630–634. 44. Brow, D. A., & Guthrie, C. (1990). Transcription of a yeast U6 snRNA gene requires a polymerase III promoter element in a novel position. Genes & Development, 4(8), 1345-1356. 45. Wei, Y., Qiu, Y., Chen, Y., Liu, G., Zhang, Y., Xu, L., & Ding, Q. (2017). CRISPR/Cas9 with single guide RNA expression driven by small tRNA promoters showed reduced editing efficiency compared to a U6 promoter. RNA, 23(1), 1-5. 46. Guthrie, C., & Patterson, B. (1988). Spliceosomal snRNAs. Annual review of genetics, 22(1), 387-419. 47. Brow, David A., & Christine Guthrie. (1988). Spliceosomal RNA U6 is remarkably conserved from yeast to mammals. Nature. 334(21) 213-218. 48. Lubliner, S., Keren, L., & Segal, E. (2013). Sequence features of yeast and human core promoters that are predictive of maximal promoter activity. Nucleic acids research, 41(11), 5569-5581. 49. Lubliner, S., Regev, I., Lotan-Pompan, M., Edelheit, S., Weinberger, A., & Segal, E. (2015). Core promoter sequence in yeast is a major determinant of expression level. Genome research, 25(7), 1008-1017. 50. Weninger, A., Hatzl, A. M., Schmid, C., Vogl, T., & Glieder, A. (2016). Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris. Journal of biotechnology, 235, 139-149. 51. Weninger, A., Glieder, A., & Vogl, T. (2015). A toolbox of endogenous and heterologous nuclear localization sequences for the methylotrophic yeast Pichia pastoris. FEMS yeast research, 15(7). 52. Jakočiūnas, T., Jensen, M. K., & Keasling, J. D. (2016). CRISPR/Cas9 advances engineering of microbial cell factories. Metabolic engineering, 34, 44-59. 53. Severinov, K., Ispolatov, I., & Semenova, E. (2016). The influence of copy-number of targeted extrachromosomal genetic elements on the outcome of CRISPR-Cas defense. Frontiers in Molecular Biosciences, 3. 54. Caitlin S. (2017). Editing the editor: Genome editing gets a makeover with CRISPR 2.0. Science, 355, 210. 55. Vanegas, K. G., Lehka, B. J., & Mortensen, U. H. (2017). SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae. Microbial cell factories, 16(1), 25. 56. Krooth, R. S., Hsiao, W. L., & Potvin, B. W. (1979). Resistance to 5-fluoroorotic acid and pyrimidine auxotrophy: a new bidirectional selective system for mammalian cells. Somatic Cell and Molecular Genetics, 5(5), 551-569. 57. Abad, S., Kitz, K., Hörmann, A., Schreiner, U., Hartner, F. S., & Glieder, A. (2010). Real‐time PCR‐based determination of gene copy numbers in Pichia pastoris. Biotechnology journal, 5(4), 413-420. 58. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., & Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121), 819-823. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/2416 | - |
| dc.description.abstract | 基因編輯 (Genome editing) 技術近年來迅速發展,可以針對特定位點進行突 變、基因插入或刪除及基因置換等編輯。基因編輯原理簡單,幾乎可以應用在任 何物種,不僅加速學術研究,更可用於研發市場導向的產品。其中,Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) 更是蓬勃發展。相較於其他基因編輯技術,CRISPR/Cas9 因設計簡單、編 輯效率高,雖然近五年才開始發展但已累積大量的研究資料。CRISPR/Cas9 除了 基因編輯的功能,更進一步發展出如 CRISPRi、CRISPR display、 CRISPR screen 等應用,CRISPR/Cas9 的前景不可限量。
Pichia pastoris 為常用的異源基因表現物種。 P. pastoris 表現異源蛋白質具 有可嚴謹調控、大量生產等優點,但由於其同源重組 (homologous recombination, HR) 仰賴限制酶的專一性序列,本身又缺少 RNA 干擾 (RNAi) 機制,是 P. pastoris 表達系統缺點之一。前人曾在 P. pastoris 建立 CRISPR/Cas9 系統編輯 內生性基因,並以送入異源基因證實 CRISPR/Cas9 系統可增進同源重組效率。 然而此系統表現 single guide RNA (sgRNA) 時須額外預測其結構及序列,使得設 計上較為複雜,欲在 P. pastoris 方便應用 CRISPR/Cas9 系統仍有改善空間。 本研究欲從 P. pastoris 選殖其內生性 U6 基因的啟動子,並以此啟動子表 現 CRISPR/Cas9 系統之 sgRNA。預期透過將異源蛋白質基因點突變以證明其可 用性。此 CRISPR/Cas9 系統在 P. pastoris 中的建立,將使 CRISPR/Cas9 的設 計更簡單,除了提升轉形效率,未來更可將 CRISPR/Cas9 系統延伸應用,例如 與轉錄因子結合,使 P. pastoris 成為更有效率的應用平台。 | zh_TW |
| dc.description.abstract | Genome editing technique was fast developing recently. It allowed researchers to edit genes through point mutation, insertion, deletion or replacement. The principle of genome editing was so simple that it could be applied in generally all species. It accelerated not only the development of science, but also the product related to people’s life. Among the genome editing techniques, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) was the most popular one. CRISPR/Cas9 was easy to design and was efficient in editing. Many publish researches were accumulated since it developed five years ago. Moreover, there were many techniques related to CRISPR developed like CRISPRi, CRISPR display, CRISPR screen, and so on.
Pichia pastoris was widely used to express exogenous proteins. It had many advantages like strictly controlled and large-scale expression. However, while performing homologous recombination, the target sequence was restricted to the restriction enzymes used. Also, there was no RNA interference (RNAi) system in P. pastoris. Therefore, there was still room for improvement for P. pastoris as protein expression platform. Researchers had established CRISPR/Cas9 system, edited endogenous genes and sent template into P. pastoris to prove that CRISPR/Cas9 could increase the HR efficiency. However, the sequence and structure of the single guide RNA (sgRNA) in the system should be calculated additionally. The design of CRISPR/Cas9 system in P. pastoris could be simplified for application. This thesis aimed to find the endogenous U6 promoter from P. pastoris and use U6 promoter to express sgRNA. The concept was proved by the point mutation of genes of interest. The establishment of the system could make the application of CRISPR/Cas9 in P. pastoris easier. It not only could improve the efficiency of transformation, but also could perform other techniques related to CRISPR/Cas9 like the combination with transcription factors. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-13T06:39:58Z (GMT). No. of bitstreams: 1 ntu-106-R04b22019-1.pdf: 2752294 bytes, checksum: 437074d2c128d798a187505c1f57393a (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 謝誌 I
摘要 III Abstract IV 目錄 VI 圖目錄 IX 表目錄 X 附圖目錄 XI 第一章、前言 1 一、基因編輯 (Genome editing) 1 1. 簡介 1 2. 常見技術 1 3. 應用 2 二、Clustered regularly Interspaced short palindromic repeats (CRISPR)/CRISPR- associated protein 9 (Cas9) 4 1. CRISPR 簡史 4 2. CRISPR 組成 4 3. CRISPR 機制 5 4. CRISPR 應用與發展 5 5. CRISPR 之挑戰 7 三、Pichia pastoris 8 1. 微生物蛋白質表現系統 8 2. Pichia pastoris 9 3. 啟動子選擇 9 四、RNA 聚合酶與啟動子 11 1. RNA 聚合酶 II 11 2. RNA 聚合酶 III 11 3. U6 啟動子 12 四、研究動機與目的 13 五、本篇論文具體目標 14 六、本篇論文架構 15 第二章、材料與方法 16 一、菌株及培養條件 16 1. Escherichia coli 16 2. Pichia pastoris 16 二、培養基及製備方法 16 三、質體建構 21 1. pGAPZA-EGFP 表現質體 21 2. pPIC3.5K-Cas9-U6 啟動子-sgRNA 表現質體 21 四、Pichia pastoris 轉形 28 1. 質體製備 28 2. Pichia pastoris 勝任細胞製備 28 3. Pichia pastoris 電轉 28 4. 轉形株篩選 29 五、sgRNA 設計並以 U6 啟動子轉錄 sgRNA 30 六、EGFP 拷貝數確認 32 七、Cas9 蛋白質表現 33 1. 誘導表現 33 2. 破菌 33 3. 聚丙烯醯胺膠體電泳 (SDS-PAGE) 33 4. 免疫染色 37 八、螢光偵測 38 1. 流式細胞儀 38 2. 螢光顯微鏡 38 第三章、結果 39 一、U6 啟動子序列獲得 39 二、Pichia pastoris 轉形株篩選與確認 42 三、U6 啟動子轉錄功能確認 45 四、EGFP 拷貝數確認 48 五、Cas9 蛋白質表現 49 六、螢光偵測 55 1. 流式細胞儀 55 2. 螢光顯微鏡 55 第四章、討論 61 一、以 GAP 啟動子啟動單一拷貝數 EGFP 作為標的之優劣 61 二、sgRNA 之使用 62 三、U6 啟動子之序列長度 63 四、CRISPR 系統之植入 64 五、5-FOA plate 之實驗結果 65 六、sgRNA、Cas9 蛋白質之表現量 66 七、Pichia pastoris CRISPR 系統之未來發展 67 第五章、結論 69 第六章、參考文獻 70 | |
| dc.language.iso | zh-TW | |
| dc.subject | U6 啟動子 | zh_TW |
| dc.subject | 基因編輯 | zh_TW |
| dc.subject | CRISPR | zh_TW |
| dc.subject | CRISPR | en |
| dc.subject | genome editing | en |
| dc.subject | U6 promoter | en |
| dc.title | 以內生性 U6 啟動子發展 Pichia pastoris CRISPR/Cas9 系統 | zh_TW |
| dc.title | Development of CRISPR/Cas9 system in Pichia pastoris
using endogenous U6 promoter | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳?承(Hsuan-Chen Wu),凌嘉鴻(Steven Lin),陳浩仁(Hau-Ren Chen),林晉玄(Ching-Hsuan Lin) | |
| dc.subject.keyword | 基因編輯,CRISPR,U6 啟動子, | zh_TW |
| dc.subject.keyword | genome editing,CRISPR,U6 promoter, | en |
| dc.relation.page | 78 | |
| dc.identifier.doi | 10.6342/NTU201702301 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2017-08-01 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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