中國倉鼠卵巢細胞(CHO cell)在蛋白藥市場中扮演相當重要之角色，能夠有效的表現出與哺乳動物類似的複雜結構蛋白，在dhfr基因缺陷的CHO cell內，可利用外源dhfr基因與重組蛋白基因建構於相近的位置，利用額外添加DHFR抑制劑methotrexate (MTX)的方式，迫使CHO cell進行dhfr基因擴增，同時一併也會增加重組蛋白基因的基因套數，利用此方式達到提高重組蛋白表現。
第一部分研究利用CRISPR/Cas9系統衍伸出的CRISPRi系統能有效利用專一性的gRNA進行基因調控，利用CRISPRi抑制外源dhfr基因進而增加基因擴增時的篩選強度，進而提升基因擴增後重組蛋白基因之表現量。本實驗以報導基因EGFP作為重組目標蛋白，相較於傳統方式，CRISPRi抑制dhfr基因只需以250 nM濃度之MTX即可有效提升蛋白表現量。
本研究證實利用CRISPRi抑制dhfr基因進而增加基因擴增時篩選強度，EGFP表現量能增加約3倍，基因套數增加約3.6倍。此外，我們利用CRISPRi技術應用於生產Granulocyte colony stimulating factor (G-CSF)蛋白，結果顯示利用CRISPRi組能提升蛋白產量約2.3倍，研究結果證明利用此方式不只能增加報導基因EGFP蛋白產量也能實際應用於高商業價值之蛋白藥物生產。
第二部分則是利用新型的CRISPR/Cas13d進行CHO cell engineering，CRISPR/Cas13d能專一性的切割目標RNA進行基因調控，我們利用CRISPR/Cas13d抑制CHO cell engineering與相關之基因bak、bax及pdk1 (pyruvate dehydrogenase kinase 1)，並以EGFP作為重組目標蛋白，在以CRIPSR/Cas13d持續抑制bak、bax及pdk1之下，能有效提升EGFP表現量達1.5倍。在此驗證CRISPR/Cas13d系統應用於CHO cell engineering之可行性。

CHO cell is an important host for biopharmaceutical production. Generation of stable CHO cell typically requires co-integration of dhfr and foreign gene into chromosomes and subsequent methotrexate (MTX) selection for co-amplification of dhfr and foreign gene. CRISPR interference (CRISPRi) is an emerging system that effectively suppresses gene transcription through the coordination of dCas9 protein and guide RNA (gRNA). However, CRISPRi has yet to be exploited in CHO cells. In the first part we used CRISPRi system to suppress dhfr transcription in CHO cells. We next generated stable CHO cell clones co-expressing DHFR, the model protein (EGFP), dCas9 and gRNA targeting dhfr. Combined with MTX selection, CRISPRi-mediated repression of dhfr imparted extra selective pressure to force CHO cells to co-amplify more copies of dhfr and egfp genes. The CRISPRi approach increased the dhfr copy number for ≈3-fold, egfp copy number for ≈3.6-fold and enhanced the EGFP expression for ≈3.8-fold, without impeding the cell growth. Furthermore, the strategy enhanced the productivity of granulocyte colony stimulating factor (G-CSF) for ≈2.3-fold. Our data demonstrate, for the first time, the application of CRISPRi in CHO cells to enhance recombinant protein production.
In the second part we used a novel tool CRISPR/Cas13d to specifically regulate CHO gene. We demonstrated that CRISPR/Cas13d can multiplexingly suppress Bak, Bax and PDK1 (Pyruvate dehydrogenase kinase 1) gene. As a result, the EGFP expressing CHO cells can effectively increase EGFP expression ≈1.5-fold without interfering with cell growth, thus underscoring the potential of CRISPR/Cas13d in CHO cell engineering.

第一章 文獻回顧 1
1-1 中國倉鼠卵巢細胞表現系統 1
1-1-1 外源基因在中國倉鼠卵巢細胞中之表現 1
1-1-2 中國倉鼠卵巢細胞表現系統 3
1-1-3 中國倉鼠卵巢細胞之細胞工程 4
1-1-4 中國倉鼠卵巢細胞之醣基化作用 5
1-2 CRISPR系統 7
1-3 CRISPRi應用與發展現況 9
1-4 CRISPR/Cas13系統 10
1-5 CRISPR/Cas13應用與發展現況 12
1-6研究動機 13
第二章 材料與方法 15
2-1 CRISPRi表現載體之建構 15
2-1-1 dCas9-KRAB表現載體建構 15
2-1-2 pCRISPRi表現載體建構 15
2-1-3 pDHFR-P2A-EGFP表現載體建構 16
2-2 CRISPRi抑制DHFR基因效率測試 16
2-3 EGFP-DHFR基因擴增測試之表現載體建構 16
2-4 建立穩定表現EGFP及DHFR基因之細胞株挑選 17
2-5細胞單株化 17
2-6 建立穩定表現CRISPRi基因之細胞群及篩選 18
2-7 以MTX篩選進行EGFP-DHFR基因擴增 19
2-7-1 CRISPRi抑制EGFP螢光強度之絕對定量 19
2-7-2 qRT-PCR進行egfp及dhfr mRNA之相對定量 20
2-7-3 Q-PCR進行Genomic DNA gene copy number之絕對定量 20
2-8 CRISPRi對於CHO細胞生長速度影響之測試 20
2-9 蛋白藥物G-CSF-DHFR基因擴增之表現載體建構 21
2-10 穩定表現G-SF及DHFR基因之細胞株挑選 21
2-11 穩定表現CRISPRi基因之細胞株篩選 22
2-12 以MTX篩選進行G-CSF-DHFR基因擴增 22
2-12-1 G-CSF蛋白產量之絕對定量 22
2-12-2 以qRT-PCR進行 gcsf mRNA之相對定量 23
2-12-3 Q-PCR進行Genomic DNA copy number之絕對定量 23
2-12-4 西方墨點法偵測G-CSF蛋白表現量 23
2-13 pEIF1-Cas13dPZgRNA表現載體建構 24
2-14 pIS-Cas13dPZgRNA表現載體建構 24
2-15 CRISPR/Cas13d抑制外源EGFP效率測試 25
2-16 CRISPR/Cas13d抑制內源性EGFP效率測試 25
2-17 穩定表現CRISPR/Cas13d抑制內源性EGFP效率測試 25
2-18 CRISPR/Cas13系統抑制CHO-K1細胞內源性基因測試 26
2-19 穩定表現CRISPR/Cas13系統抑制CHO-K1細胞源性基因 26
2-20 以CRIPSR/Cas13調控內源性基因提EGFP蛋白產量 26
2-20-1 穩定表現CRISPR/Cas13及EGFP之細胞株建立 26
2-20-2 CRISPR/Cas13調控PDK1、Bax、Bak提高EGFP表現量 26
2-21 CRISPR/Cas13系統影響細胞生長速度測試 27
2-22 搭配Transposase system提高外源基因重組效率 27
2-23 以CRIPSR/Cas13調控內源性基因提IgG蛋白產量 27
2-23-1 DG44 IgG細胞轉染條件測試 27
2-23-2 Cas13抑制Bak、Bax及PDK1之DG44IgG細胞群建立 28
2-23-3 表現CRISPR/Cas13d之DG44IgG批次發酵 28
第三章 實驗結果 CRISPRi提高蛋白產量 29
3-1 CRISPRi抑制DHFR基因效率測試 29
3-2 建立穩定表現EGFP、DHFR及CRISPRi之細胞株挑選 29
3-3 CRISPRi抑制dhfr能有效提升重組蛋白表現 31
3-4 CRISPRi抑制dhfr能有效提升增加基因擴增效果 32
3-5 CRISPRi抑制dhfr基因不影響細胞生長速度 32
3-6 利用CRISPRi抑制dhfr之方法能增加G-CSF蛋白產量 33
3-7 CRISPR/Cas13抑制外源報導基因EGFP 34
3-8 穩定表現CRISPR/Cas13可持續抑制內源性報導基因 35
3-9 CRISPR/Cas13d抑制CHO-K1內源性基因 36
3-10 以CRISPR/Cas13d同時抑制Bak, Bax及PDK1增加蛋白表現 37
3-11 在CHOEGFP細胞內額外表現Cas13d不影響細胞生長速度 38
3-12 Transposase system提升外源基因重組效率 38
3-13 Transposase system增加CRISPR/Cas13d抑制基因能力 39
3-14 CHO DG44 IgG細胞轉染方式建立 39
3-15 CRISPR/Cas13d在CHO DG44IgG細胞內抑制基因效率 40
第四章 討論 62
第五章 未來展望 68
5-1 利用CRISPR/Cas13d提升CHO重組蛋白之質量 68
5-2 利用CRISPR/Cas13d提高IgG蛋白產量 69
第六章 發表專利及期刊 70
第七章 參考文獻 89

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