CRISPR/Cas系統是在原核生物中發現的防禦機制，主要作用為利用CRISPR RNA為標的識別位點、Cas蛋白為核酸酶以幫助生物體偵測入侵者的核酸並將其分解。這套系統目前已被發展為有效的基因編輯技術且被廣泛應用於分子生物學領域，尤其是在基因工程領域中。CRISPR/CasF系統是一種主要發現於大型噬菌體中的新型CRISPR/Cas系統，除了不是在細菌等原核生物中所發現這項特點之外，CasF蛋白的體積只有最常被使用的Cas9蛋白體積的大約一半，由此衍伸出在運送上的方便性使其被預期可能有機會激發CRISPR/Cas系統的進一步應用。在這項實驗計畫中，我們嘗試通過不同的方式建立在酵母菌中表現CasF-2的機制並針對其DNA編輯能力進行後續測試。首先我們利用基因序列優化(codon optimization)的方式將符合酵母菌基因表現偏好的CasF-2序列建構至質體中，再藉由質體轉送和基因嵌入等兩種方式將序列送入菌內，期望能夠在模式生物酵母菌中建立起透過質體表現和透過基因表現等兩種不同的CasF-2蛋白表現方式。接著我們進行了西方點墨法實驗以測定CasF-2蛋白質的表現量，並從實驗結果中觀察到在培養溫度介於20℃至30℃之間時，溫度越低CasF-2蛋白的表現量有越高的趨勢。在接下來針對CasF-2的基因編輯能力測試中我們選擇使用生存率試驗和重組增強率試驗來進行評估，然而兩項試驗結果在student’s t-test的分析下皆顯示出無論是對菌種還是培養溫度進行調整都沒有任何一組的CasF-2有完成DNA切割的跡象，反觀對照組的Cas9則表現出了很強的切割能力。後續的蛋白質位置分析實驗結果中可看出CasF-2在細胞中的所在位置與細胞核相互對應，由此推測CasF-2應確實有進入細胞核。從我們的實驗成果發現到酵母菌中所表現的CasF-2於20°C至30°C的溫度範圍內隨著培養溫度的降低蛋白質表現量有升高的趨勢，且CasF-2在細胞中與細胞核的所在位置相吻合，可推論出在蛋白質表現與進入細胞核的過程中並無異常，然而整體CRISPR/CasF-2系統卻沒有呈現出有效的DNA切割成果。綜上所述，CRISPR/CasF系統在酵母菌中是否能夠發揮作用的關鍵因素仍然需要更進一步的研究探索。

CRISPR/Cas systems are defense mechanisms found in prokaryotes that give them the ability to target foreign nucleic acids and degrade them. The systems with CRISPR RNA for recognition of the target site and Cas protein as the nuclease have been widely used in the field of molecular biology, especially in gene engineering. CRISPR/CasF system was a relatively new type of CRISPR/Cas system, which was found in Biggiephage rather than bacteria. The small size of CasF protein, about half the mass of the most commonly used CRISPR-Cas9, might be able to inspire further CRISPR-Cas system applications. Here we tried to build up the newly found CRISPR/CasF system in the model organism S. cerevisiae. The codon optimized CasF-2 sequence was constructed into plasmids and CasF-2 was expressed either from a plasmid by plasmid transformation or from a chromosomal location by genome insertion. Western blot was conducted to examine the expression of CasF-2 and a tendency of higher protein expression level with lower culture temperature from 20°C to 30°C was observed. We utilized two assays, viability assay and recombination enhancement assay, to examine the cleavage of target DNA. However, there wasn’t any sign of target DNA cleavage by CasF-2 in any strain under any temperature tested by the two assays, while Cas9, which serves as a comparing group, appeared to have strong cutting performance. We further examined the cellular location of CasF-2 and found it corresponded to the site of cell nucleus, proving the entry of CasF-2 into the nucleus. In conclusion, our data demonstrate that CasF-2 appeared to have the expression tendency of higher expression level under lower culture temperature within the range 20°C to 30°C in S. cerevisiae. The CRISPR/CasF-2 system we constructed showed poor cutting in S. cerevisiae although the expression and cellular location of CasF-2 seem normal. The key factors for CRISPR/CasF system to work in S. cerevisiae still remain to be investigated.

中文摘要 I
Abstract II
致謝辭 III
Table of contents IV
List of Tables V
List of Figures VI
1.Introduction 1
2.Materials and Methods 4
2.1. Plasmid Construction 4
2.2. Yeast Strain Construction 4
2.3. Ellis Lab CRISPR/Cas9 Tools 8
2.4. Western Blot analysis 8
2.5. Viability Assay 9
2.6. Recombination Enhancement Assay 9
2.7. Green Fluorescent Protein Detection 10
3. Results 11
3.1. Construction of CasF-2 expressing system in S. cerevisiae 11
3.1.1. Construction of the CasF-2 expressing plasmid p416TEF_CasF-2 11
3.1.2. Transformation of CasF-2 locus into S. cerevisiae 11
3.2. Confirmation of CasF-2 Expression in S. cerevisiae 12
3.3. Construction of CasF-2 sgRNA Expressing Plasmids 15
3.4. Assessment of CasF-2 genome editing ability 17
3.4.1. Cutting Efficiency Test with sgRNA targeting Leu2 locus 17
3.4.2. Recombination Enhancement Test with sgRNA targeting Leu2 locus 23
3.4.3. EGFP Cutting Efficiency Test 27
3.4.4. Examination of CasF-2 Cutting Ability with Normalization 33
3.5. CasF-2 Location Detection 36
4. Discussion 39
5. References 43
6. Appendix 45

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