利用超早熟水稻Kitaake結合簡單的突變篩選方法建立高效率CRISPR/Cas9基因編輯技術

Efficient CRISPR/Cas9-mediated Genome Editing in Rice Using an Early Flowering Cultivar Kitaake Combined with Simple Mutant Screening Approaches

林德哲(Te-Che Lin) ； 侯雅文(Ya-Wen Hou) ； 吳涵䭲(Han-Yi Wu) ； 蔡育彰(Yu-Chang Tsai)

《作物、環境與生物資訊》 14卷3期 (2017/09) Pp. 175-186

https://doi.org/10.30061/CEB.201709_14(3).0004

摘要

水稻為目前單子葉植物功能性基因體研究重要的模式植物，為加速水稻功能性基因體研究，本研究利用由播種至開花平均僅52生育日的超早熟水稻Kitaake品種，並以CRISPR/Cas9基因編輯技術針對目標序列或區間進行編輯。經過農桿菌轉殖的轉殖株除先經過抗生素Hygromycin篩選帶有CRISPR/Cas9載體之再生Kitaake植株。本試驗中並以DNA原態聚丙烯醯胺膠體電泳(PAGE)分析、高解析熔點分析(HRM)以及測序等不同方法來篩選鑑定編輯後之基因序列，結果顯示所使用的載體成功的對目標基因進行定向編輯，在轉殖植株數目較少的T_0世代可精確地以原態PAGE分析編輯序列，再以測序確認序列之變異型式，而在植株數量較多的T_1及後續分離或回交世代，則可同時以原態PAGE以及HRM分析方式進行檢測，相較於原態PAGE，HRM方式可快速篩選大量的樣本，並具有很高之準確度。此分析方法可快速並有效率的建立突變水稻品系作為特定基因或序列之功能探討。

關鍵字

基因編輯 ； CRISPR/Cas9 ； 水稻 ； 高解析熔點分析 ； Kitaake

並列摘要

Rice is an important model plant for studying gene function in monocotyledonous plants. In order to speed up functional genomics studies in rice, an extremely early flowering rice cultivar, Kitaake, was used in this study. The average flowering time for Kitaake in the greenhouse is around 52 days in both spring and fall in North Taiwan. To target specific genes or chromosome regions, CRISPR/Cas9 technology was employed. After Agrobacterium-mediated transformation, putative transgenic plants were screened by hygromycin. Three methods were conducted to determine the mutation frequency including native-PAGE, high-resolution melting (HRM) and sequencing. In T0 generation, genome-edited (GE) plants can be accurately determined by two PCR-based approaches, native-PAGE and sequencing assay. In later generations, HRM and native-PAGE assays can be employed for high efficient and high throughput mutant screening. In comparison to native-PAGE, HRM assay was able to screen GE plants with a higher speed, throughput and accuracy. Through this system, the function of specific genes or genomic region in rice can be explored quickly and efficiently.

並列關鍵字

Gene editing ； CRISPR/Cas9 ； Rice ； High resolution melting ； Kitaake