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標題: | 以CRISPR系統編輯NK-92細胞基因體 CRISPR-mediated Genome Editing of NK-92 |
作者: | Rih-Sheng Huang 黃日昇 |
指導教授: | 凌嘉鴻(Steven Lin) |
關鍵字: | CRISPR-Cas9,NK-92,免疫治療,基因編輯, CRISPR-Cas9,NK-92,Immunotherapy,Genome editing, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 自然殺手細胞是一種在免疫系統中可有效針對惡性腫瘤細胞進行毒殺的免疫細胞,在免疫治療中被視為具有相當潛力的細胞種類。自然殺手細胞的活化涉及一系列受體與目標細胞的配體互動所組成的複雜程序,該程序由活化型以及抑制型受體傳遞的訊息所控制並最終決定自然殺手細胞活化與否。精準且穩健的遺傳學工具可以有效地解析活化過程中的關鍵步驟與機制並可運用於強化自然殺手細胞針對惡性腫瘤細胞的毒殺能力。目前,廣泛運用於自然殺手細胞的遺傳學工具主要是慢病毒感染技術,然而該技術不僅耗時、低效率且具有隨機整合的風險。此論文旨在開發運用於自然殺手細胞之CRISPR-Cas9基因編輯平台。透過電轉染組裝好之Cas9:guide RNA核蛋白,NK-92細胞株的基因剔除效率在經過一系列的條件優化後可高達90%並同時維持80%細胞存活率。Cas9核蛋白方法亦可將attP39B重組序列精準插入細胞基因體特定位置中,該序列為phiC31重組酵素導引的基因重組平台。本研究建立了一個NK-92細胞株的基因編緝步驟準則,未來可應用於人類初代自然殺手細胞基因編輯以加速生物科學研究與醫療工程開發。 Natural Killer (NK) cell is an effective arsenal in immune system against malignant cells, and is highly regarded as a promising cell type for immunotherapy. Activation of NK cell is an intricate process that involves the interaction between a series of NK cell surface receptors and target cell ligands, providing positive and negative signaling to ultimately determine the activation status of NK cells. A robust and precise genetic tool will be useful to dissect this complex mechanism and to allow engineering of NK cells with enhanced cytotoxicity against malignant cells. Currently, the genetic modification of NK cells relies largely on retroviral transduction, which is time-consuming, inefficient and unsafe due to random viral integration. This thesis aimed to develop a CRISPR-Cas9 gene editing platform for NK cells. By electroporation of pre-assembled Cas9:guide RNA ribonucleoprotein (Cas9 RNP) complexes, the gene knockout process in NK-92 cell line was optimized to achieve editing efficiency up to 90% while maintaining the viability up to 80%. Cas9 RNP method also enabled site-specific knock-in of attP39B sequence, a common landing pad for phiC31-mediated recombination. Collectively, this thesis established a protocol for NK-92 genome editing that can be adopted for primary NK cells to facilitate biological research and therapeutic engineering. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74510 |
DOI: | 10.6342/NTU201902538 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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