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標題: | 以內生性 U6 啟動子發展 Pichia pastoris CRISPR/Cas9 系統 Development of CRISPR/Cas9 system in Pichia pastoris using endogenous U6 promoter |
作者: | Meng-Hsi Tsai 蔡孟羲 |
指導教授: | 黃慶璨(Ching-Tsan Huang) |
關鍵字: | 基因編輯,CRISPR,U6 啟動子, genome editing,CRISPR,U6 promoter, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/2416 |
DOI: | 10.6342/NTU201702301 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 生化科技學系 |
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