CRISPR/Cas9基因組編輯技術於作物改良之應用現況

Current Applications of CRISPR/Cas9 Genome Editing Technology on Crop Improvement

蘇奕臣(Yi-Chen Su) ； 黃鵬林(Pung-Ling Huang) ； 杜宜殷(Yi-Yin Do)

《臺灣園藝》 62卷4期 (2016/12) Pp. 201-212

摘要

基因組定點編輯技術在近十年來受到科學家的重視。除了似轉錄活化蛋白效應子核酸酶(Transcription activator-like effector nucleases, TALEN) 及鋅指核酸酶(zincfinger nuclease, ZFN)常用的基因組編輯工具外，群聚且規律性間隔的短廻文重複序列(clustered regularly interspaced short palindromic repeats, CRISPR)/CRISPR 關聯蛋白質9(CRISPR-associated protein 9, Cas9)是更快速且便宜的新選項。CRISPR/Cas9 源自於原核生物的第二型CRISPR/Cas 防禦系統，經人為改良後，核酸內切酶Cas9 與單一導引RNA(single-guide RNA, sgRNA)結合所形成的複合物，能辨識目標基因組DNA 上的前間隔序列鄰近構形(protospacer adjacent motif, PAM)，使目標基因組DNA 產生雙股斷裂(doublestrand break, DSB)，再經過非同源染色體末端連接(non-homologous end-joining, NHEJ)或同源重組(homologous recombination)，可達到基因編輯的效果。目前已透過CRISPR/Cas9改變多項農作物性狀，如花色調控、延長櫥架壽命、抗殺草劑、提升抗逆境能力、增加抗病性等。利用CRISPR/Cas9 創造的作物透過孟德爾遺傳分離後，不含其他生物的外源基因，因此不受基因改造法規的限制，成為科學界研究與開發的最新利器。

關鍵字

同源重組 ； 非同源染色體末端連接 ； 前間隔序列鄰近構形 ； 單一導引RNA

並列摘要

In the recent decade, scientists put more attention on genome site-specific editing technologies, such as transcription activator-like effector nucleases (TALEN) and zinc finger nuclease (ZFN). Recently, a new 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 targeted sequence will form a complex with the endonuclease Cas9 to recognize the protospacer adjacent motif (PAM) near the target sequence. Then the target sequence is edited by non-homologous end-joining (NHEJ) or homologous recombination. So far, several agricultural traits has been manipulated by CRISPR/Cas9, including change of flower color, extension of shelf-life, development of herbicide-resistant crops, and improvement of stress- and disease-resistance of crops. After Mendelian segregation the CRISPR/Cas9-edited plant without any foreign genes is not classified as a genetic modified organism. Due to the ease of operation and less regulation of genetically modified organisms, CRISPR/Cas9 becomes a powerful tool to for plant breeders and researchers.

並列關鍵字

homologous recombination ； non-homologous end joining ； protospacer adjacent motif ； single-guide RNA