植物CRISPR/Cas9多基因编辑载体构建和突变分析的操作方法

A protocol for CRISPR/Cas9-based multi-gene editing and sequence decoding of mutant sites in plants

CRISPR/Cas9基因组编辑技术是植物基因功能研究与作物改良的有效工具.为此,本实验室开发了高效的CRISPR/Cas9植物多基因编辑载体系统.本载体系统包括6个双元载体和12个含有不同U3/U6启动子的sg RNA中间载体,可满足对单子叶和双子叶植物的遗传转化以及不同抗生素筛选的要求,具有简便、高效,可同时对多基因进行编辑的特点.此外,为了能更高效地应用基因组编辑技术,还开发了一站式在线分析工具包CRISPR-GE.为方便研究人员利用CRISPR/Cas9系统进行植物基因组编辑,本文提供了从靶点选择、CRISPR/Cas9多靶点双元载体构建,以及对靶点突变序列的测序分析等详细的操作方法,以及常见的问题解答.

Genome editing with CRISPR/Cas9 has become an effective tool for gene functionality dissection and crop improvement.Accordingly, we have developed a CRISPR/Cas9 vector system for robust multi-gene editing in plants. Our inventory includes six versatile binary vectors and 12 intermediary sg RNA vectors with different U3 and U6 promoters, to achieve high efficient multi-gene editing, under specific antibiotic selections in both monocot and dicot plants. Additionally, we also have developed an integrated webbased software toolkit, CRISPR-GE, to facilitate the utilization CRISPR/Cas9 in plants. In this protocol, we provide detailed procedures for using the CRISPR/Cas9 system, including target site selection, construction of CRISPR/Cas9 binary vectors, Sangersequencing-based genotyping of mutant sites, and troubleshooting for common problems.