摘要
基因體編輯技術(genome editing technology)因可準確改變基因體中特定位點的核苷酸序列，近年來成為廣泛應用於植物品種改良及植物生理研究的策略，其中又以群聚且規律性間隔的短回文重複序列(clustered regularly interspaced short palindromic repeats, CRISPR)/CRISPR關聯蛋白質9 (CRISPR-associated protein 9, Cas9)為首選。運用CRISPR/Cas9系統進行基因體編輯時，首先選定欲改變的目標基因，接著分析目標基因的序列，尋找位於單個顯子內符合前間隔序列鄰近構形(protospacer adjacent motif, PAM) 5'-NGG-3'上游20個核苷酸作為導引序列(guide sequence)，置於單一導引RNA (single guide RNA, sgRNA)骨架之前端，導引序列的第一個核苷酸以G的編輯效率最佳。接著將可產生sgRNA的DNA序列構築於RNA聚合酶III啟動子如U6啟動子的下游，Cas9基因序列則由RNA聚合酶II啟動子如CaMV 35S驅動，以農桿菌媒介法或基因槍法導入植物細胞，則細胞核內目標基因的PAM上游3 bp處DNA，會發生雙股斷裂(double strand break, DSB)，再由細胞的修復系統進行非同源染色體末端連接(non-homologous end-joining, NHEJ)，插入或丟失數個核苷酸，導致目標基因喪失功能，達到剔除基因的目的。得到的基因編輯個體，再透過自交，從後代篩選僅具編輯後的目標基因、而不具gRNA及Cas9等外來序列的植株，即為不受美國農部管制的基因編輯植物，可作為育種用的親本材料。一個基因內可作為導引序列的候選區域通常不止一個，可利用暫時性表現分析系統，如原生質體聚乙二醇轉殖法(polyethylene glycol transformation)或農桿菌注入法(agroinfiltration)，先行評估各區域發生基因編輯的效率。另亦可進行試管內轉錄(in vitro transcription)產生gRNA分子、試管內轉譯(in vitro translation)生產Cas9蛋白質，於試管內組裝成核糖核酸蛋白複合物(ribonucleoprotein, RNP)，經細胞核轉染(transfection)，編輯效果更佳。得到的基因編輯細胞或植株，經抽取基因組DNA，以高解析熔解曲線分析(High resolution melting curve analysis, HRMA)、T7 endonuclease I或Surveyor nuclease assay、聚合酶連鎖反應(polymerase chain reaction)、DNA定序等方法檢測編輯結果。CRISPR/Cas9系統除了運用於剔除基因的功能，尚可進行基因片段的插入、單一核苷酸的置換、染色體的表觀結構分析、染色體影像分析、順式作用元件(cis-acting element)修飾等。
並列摘要
Genome editing technologies can be effectively employed to modify the plant genome by precise alteration in DNA sequences. Recently, genome site-specific editing technologies have been applied on plant breeding and physiology studies. The method based on bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) type II prokaryotic adaptive immune system has become a novel tool for genome site-specific editing technology. An engineered single-guide RNA (sgRNA) that specifies a 20 bp targeted sequence driven by a RNA polymerase promoter will form a complex with the endonuclease Cas9 to recognize the protospacer adjacent motif (PAM) near the target sequence. Then Cas9 trigger double-strand breakage of DNA at 3 bp upstream to PAM and the target sequence is edited by non-homologous end-joining (NHEJ) or homologous recombination. Deletion, insertion, or base substitution may occur at the breakage site. After Mendelian segregation the CRISPR/ Cas9-edited plant without any foreign genes is not classified as a genetic modified organism. Protoplast transfection, leaf agroinfiltration, and Agrobacterium-mediated transformation can be used to deliver plasmid containing sgRNA and Cas9 construct or assembled ribonucleoprotein particle into plant cells. Gene editing events can be detected by several methods, such as high resolution melting curve analysis (HRMA), T7 endonuclease I or Surveyor nuclease assay, polymerase chain reaction, and DNA sequencing. Not only gene knockout but also gene insertion, single base substitution, epigenetic modulation, genomic imaging, and modification of cis-acting element can be achieved by CRISPR/Cas9 editing system.