通过优化gRNA设计提高CRISPR/Cas9系统中基因编辑效率的方法  

Methods to Improve Gene Editing Efficiency in CRISPR/Cas9 System by Optimizing gRNA

作　　者：李秋月 王小柯[1] 张亚飞 孙珍珠 王旭 江东[1] Li Qiuyue;Wang Xiaoke;Zhang Yafei;Sun Zhenzhu;Wang Xu;Jiang Dong(Citrus Research Institute of Chinese Academy of Agricultural Sciences, Chongqing, 400712)

机构地区：[1]西南大学柑桔研究所

出　　处：《分子植物育种》2019年第10期3274-3282,共9页Molecular Plant Breeding

基　　金：重庆市科技创新专项(cstc2016shmsztzx0010);江西地方特色果树种质资源创新与利用专项(S2016NYZPF-0208)共同资助

摘　　要：CRISPR/Cas9系统是一种广泛应用于动物、植物、真菌、细菌的新一代基因定点编辑技术,如何提高g RNA的编辑效率是该技术的关键点也是许多研究者关注的焦点。本研究利用CRISPR/Cas9技术对番茄miR167a前体序列进行基因编辑,根据番茄miR167a前体序列在线设计候选g RNA,同时结合体外编辑检测的方法筛选出能够在体外成功编辑的g RNA-G1,把其连接到含有荧光标记的pP1C.1C表达载体,转化番茄子叶,产生愈伤组织数116个,得到5个含有表达载体的材料,经测序发现,其中3个材料发生了编辑。结果表明,通过体外检测和在线软件设计相结合的方法来快速检测g RNA的编辑效率,能筛选出最优的g RNA用于载体的构建,与未采用体外编辑检测来设计g RNA的方法相比,显著提高了CRISPR/Cas9的编辑效率。本研究通过优化g RNA,创新了一种高效利用CRISPR/Cas9进行基因编辑的方法。CRISPR/Cas9 system is a new generation approach of gene editing technology widely used in animals,plants, fungus and bacteria. However how to improve the gene editing efficiency of g RNA is the key point of this technology. In this study, the CRISPR/Cas9 technology was used to edit the tomato miR167 a precursor sequence,firstly the candidate g RNAs was designed online according to the tomato miR167 a precursor sequence, then combined with in vitro editing and detection methods to screen out the appropriate g RNA-G1, which could be successfully edited in vitro. The g RNA-G1 was ligated to pP1 C.1 C expression vector contained the fluorescent marker, and the tomato cotyledons were transformed to produce 116 callus tissues, and five materials containing the expression vector were obtained. After sequencing three materials were successfully edited. The results show that the combination of in vitro detection and online software design can quickly improve the editing efficiency of g RNA, and the optimal g RNA can be selected out for vector construction, this method significantly improved the editing efficiency of CRISPR/Cas9 compared with the method of designing g RNA without in vitro editing. This study reported an innovated method for efficient gene editing using CRISPR/Cas9 by optimized g RNA.

关 键 词：gRNA设计 CRISPR/Cas9 体外检测 基因编辑 

分 类 号：Q78[生物学—分子生物学]