中國倉鼠卵巢細胞（CHO細胞）在生產蛋白質藥物扮演著關重要的角色。 CHO細胞具有類似人類轉譯後修飾的能力。而轉譯後修飾中的最重要的過程之一為醣基化，其中將聚醣添加到蛋白質結構中，使得修飾的蛋白質可以充分發揮作用。在該過程中存在多種醣基化酵素。我們利用調控醣基化酵素的表現量，調控聚醣基分布，從而產生具有功能性能的蛋白質藥物。因此，將能有效改善醣基化蛋白的質量，進而符合生物相似性藥物所需之特徵。最近，CRISPR / Cas13d系統已被報導能有效調控mRNA表現量，此外也相較其他工具，有較低的脫靶效應。利用CRISPR / Cas13d系統控制CHO細胞醣基化途徑的醣基化酵素表達，以產生符合生物相似性藥物的醣基分布。在這項研究中，我們構建了CRISPR / Cas13d調控FUT8基因之載體，並驗證CRISPR / Cas13d能在CHO細胞中有效降低FUT8基因表現量。研究結果顯示，在穩定表現CRISPR / Cas13d的CHO細胞中， fut8 mRNA能有效的降低表現。此外，也驗證能在CHO細胞中大量表達β-1,4半乳糖基轉移酶（β-4GalT1）以產生更多的半乳糖聚醣用於生物相似性藥物生產。最後我們驗證了CRISPR / Cas13d系統能應用於調控醣基化的可行性。

Chinese hamster ovary cells (CHO cells) play a crucial role in the commercial production of therapeutic protein drugs. CHO cells possess the capability of human-like post-translational modification. One of the most important processes involved in the post-translational modification is the glycosylation process in which the glycans are added to the protein structure so that the modified protein can fully function. There are a variety of glycosylation enzymes present in this process. We hypothesized that through controlling the expression level of the glycoenzymes, it would be able to manipulate the glycan profile, thus generate protein drugs that have enhanced performances. Therefore, the protein quality would be improved or followed bio-similarity. Recently, CRISPR/Cas13d system emerged and has become an evolution for transcriptome engineering that can efficiently inhibit the target translation of eukaryotic cells with a low frequency of the off-target effects. Utilizing CRISPR/Cas13d system to control glycosylation enzyme expression which involved in the glycosylation pathway of CHO cell was proposed to generate a glycan profile that following the biosimilarity. In this study, we constructed the vectors required for CRISPR/Cas13d-mediated inhibition of fucosyltransferase 8 (FUT8) and explored the activity of CRISPR/Cas13d in knockdown glycoenzyme. Our data showed that fut8 expression was down-regulated under the manipulation of CRISPR/Cas13d system in stable pools. Besides, β-1,4 galactosyltransferase (β4GalT1) was overexpressed in order to generate more galactose glycan for biosimilarity. Consequently, fed-batch production was further performed to study the glycan distribution as well as cell growth characteristic and protein productivity after gene regulation.

ABSTRACT 3
LIST OF FIGURE 5
LIST OF TABLE 5
Chapter 1. Literature Review 6
1.1. Introduction to Chinese hamster ovary cell (CHO cell) 6
1.2. Cellular engineering of CHO cells 8
1.3. CHO cell glycosylation 9
1.3.1. Overview of glycosylation in CHO cell 9
1.3.2. Regulation of glycosylation in CHO cell 11
1.4. CRISPR system overview 12
1.4.1. Overview of CRISPR 12
1.4.2. Type II CRISPR/Cas system and CRISPRi system 13
1.4.3. Type VI CRISPR/Cas system 13
1.4.4. CRISPR/Cas13d system 14
RESEARCH OBJECTIVE 24
Chapter 2. Experimental Method 26
2.1. Plasmid construction 26
2.1.1. Construction of pEF1a-Cas13dPZ expression plasmid 26
2.1.2. Construction of pIS-Cas13dPZ 26
2.1.3. Construction of gRNA expression cassette 27
2.1.4. Construction of β-4GalT1 overexpression plasmids 27
2.2. CHO cell culture 28
2.3. CHO cell transfection 28
2.4. Cell selection and re-adaptation 29
2.5. Serum-free fed-batch culture 29
2.6. Real-time quantitative polymerase chain reaction (qPCR) 30
Chapter 3. Experimental results 31
3.1. Suppression efficiency of CRISPRi system for fut8 endogenous gene in IgG expressed CHO-DG44 cell 31
3.2. Validation of CRISPR/Cas13d system in exogenous gene knockdown in CHO-DG44 cell 32
3.3. Utilization of CRISPR/Cas13d system in FUT8 endogenous gene transient suppression 33
3.4. Stable suppression of FUT8 by CRISPR/Cas13d system 34
3.5. Serum-free fed batch culture of engineered CHO-DG44 cells 35
3.6. Overexpression of β-4GalT1 exogenous gene in IgG expressed DG-44 cell 36
Chapter 4. Dicussion 47
Chapter 5. Future work 52
REFERRENCE 53

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