2018-08-012024-05-16https://scholars.lib.ntu.edu.tw/handle/123456789/667807摘要：CRISPR/Cas9 系統可以在定點進行 DNA 編輯，或進行基因剔除實驗，已成為一個強而有力的工具，應用於人類、阿拉伯芥、斑馬魚、小鼠、大鼠、酵母菌、線蟲、和果蠅的基因剔除。目前也已經在許多原蟲類寄生蟲發展出來，包括瘧原蟲、弓形蟲、錐蟲、和利什曼原蟲。梨形鞭毛蟲是一種單細胞原蟲的腸道病原體，寄生在小腸而引起腹瀉疾病。類似於痢疾阿米巴和其他寄生於腸道之原蟲，其生活史中的重要目的是要分化成囊體，以存活於體外並傳播疾病。梨形鞭毛蟲的生活史已被建立於試管中，因此它成為一個很重要的研究模式。在分化形成囊體時，囊壁蛋白質cwp (cyst wall protein)基因之表現增加，因此形成囊壁，但此機制目前仍不清楚。建立基因剔除(knock out)系統對梨形鞭毛蟲研究是很重要的，因梨形鞭毛蟲有四套染色體，很難利用基因刪除技術進行分子鑑定，所以發展CRISPR/Cas9 系統是迫切需要的。我們目前領先全世界其他研究此蟲的實驗室，已發展一套梨形鞭毛蟲CRISPR/Cas9 系統，可將梨形鞭毛蟲的mlf (myeloid leukemia factor)基因減量(knock down)，結果發現mlf 基因減量，可明顯減少cwp 基因表現及囊體形成，顯示MLF 蛋白質對於CWP 在囊體形成時有正向調控作用。由於目前我們的技術只能將mlf 基因減量，不能完全剔除，但以其他物種成功的例子，CRISPR/Cas9 系統的潛力仍有可能完全剔除基因。此計劃我們將以目前成功的mlf 基因為目標，設計不同載體，或加入抑制Non-homologous end joining 的藥，SCR7，以促進 homologousrecombination,希望能將mlf 基因完全剔除。也希望能設計出敲入的系統，以製造突變及加入蛋白質標籤。此技術將可套用在其他基因，對梨形鞭毛蟲的研究與發展治療用藥有莫大的幫助。<br> Abstract: CRISPR/Cas9 system has been applied for specific DNA editing or gene knock out, and used as apowerful tool for targeted gene knockout in human, Arabidopsis, zebrafish, mice, rats, yeast, nematodes, andfruit flies. CRISPR/Cas9 systems have also been developed for studies in several protozoan parasites,including Plasmodium, Toxoplasma, Trypanosoma and Leishmania. Giardia lamblia is a single cellprotozoan and also an enteric pathogen, parasitizing the small intestine and causing infectious diarrhealdiseases worldwide. Like Entamoeba and other intestinal protozoan parasites, the importance of life cycle isto differentiate into cysts to survive outside the hosts and to transmit diseases. The life cycle of Giardia hasbeen completed in vitro, making it a valuable model for research. During encystation, expression of cyst wallproteins (CWPs) increases to form cyst wall. Mechanism regulating this differentiation is still not clear. Agene knock out system is required for analyzing the role of specific molecules in vivo. The genome ofGiardia is tetraploid. It is not easy to do gene knockout for molecular characterization. Development of aclustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)system is urgent needed for studies of this organism. We are the first lab to develop the CRISPR/Cas9 systemin Giardia. Using that system, we found that knock down of the mlf gene resulted in a significant decrease ofcwp gene expression and cyst formation, suggesting a positive role of MLF in encystation. However, we canonly knock down mlf gene expression but not completely knock out mlf gene. The examples of successfulknock out of target genes in other organisms suggest the potential of CRISPR/Cas9 to knock out genes inGiardia. In this project, we will use our first successful target gene, mlf, as a model to design differentvectors for the CRISPR/Cas9 system. We will also add an inhibitor for non-homologous end joining, SCR7,to promote homologous recombination. Our aim is to knock out mlf gene completely. We also hope to designa knock in system for introduction of a mutation or a protein tag. The CRISPR/Cas9 system, allowingfunctional analysis of other genes, will be a significantly improved tool for use in Giardia studies and drug development.CRISPRCas9基因剔除梨形鞭毛蟲mlfcwp囊壁CRISPRCas9gene knock outGiardiamlfcwpcyst wall