近年來，基因編輯 (genome editing) 的研究領域與工具開發獲得了重大的突破與發展，能達到精準的基因編輯的效果。為了更有效的利用基因編輯工具，必須了解影響基因編輯與基因修復的關鍵因子，然而現行的檢驗方法相當繁瑣與耗時，主要可分為: 1. 萃取出細胞的基因體DNA，利用PCR (polymerase chain reaction, PCR) 的方法放大目標序列後，分析目標序列的突變情形； 2. 利用特殊的報導基因，並透過報導基因的表現確認編輯效果； 3. 利用桑格定序 (Sanger sequencing) 或次世代定序 (Next Generation Sequencing, NGS) 的方式直接對目標序列進行定序。在2011年時，有團隊研究出 "Traffic-Light Reporter"1，能夠分辨細胞在利用基因編輯工具產生雙股核酸斷裂 (Double-strand break, DSB) 後的修復情形; 透過流式細胞儀 (Fluorescence Activated Cell Sorter, FACS) ，觀測基因編輯後的HDR (Homology-Directed Repair, HDR) 修復的GFP訊號及NHEJ (Non-homologous end join, NHEJ) 後移碼突變 (frameshift) 產生的mCherry訊號。然而流式細胞儀檢測方法十分地耗時且必須犧牲細胞進行檢測，無法長時間追蹤基因修復後的情況。
本實驗研發出生物冷光修復報導基因(bioluminescence repair reporter, BLRR) 。利用分泌型態 (secreted type) 生物冷光酶Vargula luciferase以及Gaussia luciferase，在不犧牲細胞的情況下，簡易且快速地得知基因修復途徑，並且可以利用相同的細胞進行接續的實驗分析。透過BLRR的訊號與NGS定序的結果相比，顯示出BLRR能夠反應出隨時間變化的基因編輯與基因修復情形，且能夠檢測出最低1.23 ± 0.32% 的HDR比例，與14.7 ± 1.41% 的NHEJ比例，和一般常用的酶突變剪切法 ( Enzyme Mismatch Cleavage assay, EMC assay) 相近。同時亦能夠反應出藥物和化合物所造成的基因修復情形改變，證明BLRR為一個簡易、高成本效益、快速且靈敏的基因編輯檢測平台。

In recent year, there are major breakthroughs in the development of genome editing tools developing. Understanding the mechanism and the key factors behind genome editing could increase the performance of these tools. However, current methods for verifying genome editing results are labor intensive. These methods can be characterized into: 1) extracting genome from cells and using PCR to amplify target sequence for analyzing; 2) using a reporter gene to evaluate editing efficiency, and; 3) using sequencing methods such as Sanger sequencing or NGS to analyze target sequences. In 2011, a team developed a reporter, traffic light reporter, that can be used for determining genome editing repair by FACS. In NHEJ, the cell expressing traffic light reporter will express mCherry due to frameshift mutation; while in HDR, the cell will repair GFP sequence. Although traffic light reporter can determine genome editing outcomes easily, the requirement of collecting cells before FACS is time-consuming and does not allow high-throughput sampling.
In this research, we report a bioluminescence repair reporter, BLRR, as a new reporter platform for detecting genome editing outcomes. Using secret types of bioluminescence, Vargula luciferase and Gaussia luciferase, we are able to develop a high-throughput method to understand the genome editing results without sacrificing cells. Comparing BLRR signal with NGS result from the same groups of cells, BLRR signal can be used to observe time-dependent genome editing events and can detect as low as 1.23 ± 0.32% of HDR and 14.7 ± 1.41% of NHEJ events, same as commonly used method such as EMC assay. BLRR also can reveal the effect of the drug on genome editing events. Together, BLRR is a highly sensitive, cost-effective and high-throughput platform for genome editing events monitoring.

摘要 I
ABSTRACT II
致謝 III
目錄 IV
圖目錄 VI
表目錄 VI
縮寫表 VII
第一章、 文獻探討 1
1.1 生物冷光 (BIOLUMINESCENCE) 1
1.2基因修復 2
1.2.1 非同源性末端接合 (Nonhomologous End Joining, NHEJ) 3
1.2.2 同源重組 (Homology-Directed Repair, HDR) 6
1.3 基因編輯工具 (GENOME EDITING TOOLS) 8
1.3.1巨大限制酶 (Meganucleases) 8
1.3.2 鋅手指核酸剪切酶 (zinc finger nucleases，ZFNs) 9
1.3.3 轉錄激活樣效應因子核酸酶 (Transcription Activator-like Effector Nucleases, TALEN) 10
1.3.4常間回文重複序列叢集/常間回文重複序列叢集關聯蛋白系統 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins，CRISPR/Cas system) 12
1.4 基因編輯結果檢測 14
1.4.1 聚合反應分析方法 (PCR-based method) 14
1.4.2報導基因 (Reporter-based method) 18
1.4.3 定序分析 (Sequencing method) 19
第二章、 研究動機 26
第三章、 實驗方法 27
3.1. 實驗材料 27
3-1-1.化學藥品及試劑 27
3-1-2. 儀器 29
3.2 質體建構 31
3.3 細菌形質轉換 (BACTERIAL TRANSFORMATION) 31
3.4 限制酶剪切 32
3.5 細胞培養 32
3.6 細胞轉染 (TRANSFECTION) 33
3.7 BLRR慢病毒生產 (LENTIVIRAL PRODUCTION) 33
3.8 BLRR5穩定型細胞株 (STABLE CLONE) 建立 33
3.9 GLUC訊號強度分析 34
3.10 VLUC訊號強度分析 35
3.11 細胞活性訊號分析 35
3.12 T7E1 內切酶分析 35
3.13 TIDE 和TIDER分析 36
3.14 次世代定序分析 36
3.15 西方點墨法分析 36
第四章、 實驗結果 38
4.1 質體建構 38
4.2 BLRR 功能測驗 41
4.3 BLRR檢測極限分析 45
4.4 藥物檢測步驟優化 48
4.5 BLRR5 細胞定性測試 52
第五章、 討論 62
第六章、 參考資料 66

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