1种简单快速高效的双靶点CRISPR/Cas9载体构建方法  

A simple,fast and efficient method of constructing dual-target CRISPR/Cas9 vector

作　　者：张扬 黄维峰 周菲[1] 林拥军[1] ZHANG Yang;HUANG Weifeng;ZHOU Fei;LIN Yongjun(College of Biology Science and Technology/National Key Laboratory of Crop Genetic Improvement,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]华中农业大学生命科学技术学院/作物遗传改良国家重点实验室,武汉430070

出　　处：《华中农业大学学报》2020年第3期9-18,共10页Journal of Huazhong Agricultural University

基　　金：国家重大科技专项(2016ZX08001-001)。

摘　　要：基于Golden Gate载体构建过程,对CRISPR/Cas9双靶点敲除技术的载体构建系统进行了优化,建立了一种简单快速高效且无PCR扩增的双靶点CRISPR/Cas9载体构建方法。在本方法中,只需将设计好的2个靶点DNA小片段和1个外源片段用T4 DNA连接酶构建到CRISPR/Cas9载体上即可。用本方法对10个已报道的水稻基因构建了20个双靶点CRISPR/Cas9载体,阳性率为100%,测序结果显示无突变。该载体系统非常适合大批量CRISPR/Cas9载体的构建,同时也为其他植物中构建双靶点CRISPR/Cas9载体提供借鉴。A simple,fast and efficient method of constructing dual-target CRISPR/Cas9 vector was established based on the Golden Gate ligation.The two designed small target DNA fragments and one exogenous fragment were constructed together into the CRISPR/Cas9 vector with T4 DNA ligase.20 dual-target CRISPR/Cas9 vectors against 10 reported rice genes were constructed with this method,and the positive rate was 100%with no mutation in sequences.This vector system provides an easy,rapid and efficient method for constructing dual-target CRISPR/Cas9 vector for rice.It also provides a reference for the construction of dual-target CRISPR/Cas9 vectors in other plants.

关 键 词：水稻 CRISPR/Cas9 基因敲除 基因编辑 双靶点 载体构建 

分 类 号：Q78[生物学—分子生物学]