一种基于定量PCR的CRISPR/Cas9基因编辑作物快速检测方法的研究

A Rapid Detection Method Based on qPCR for CRISPR/Cas9 Edited Crops

基因编辑技术自诞生以来,在作物育种、基因功能分析中得到了广泛的应用,其中CRISPR/Cas9系统是目前使用最多的基因编辑技术。基于荧光定量PCR技术,针对编辑位点设计特异性探针,结合基因组快速提取方法和成熟的水稻内参基因PLD引物,对自行研发的CRISPR/Cas9系统编辑水稻的Os11N3基因进行检测,并通过合成质粒模拟不同突变情况对该方法的特异性进行了验证。研究所建立的体系可在节约时间和成本的同时获取满足qPCR检测所需的基因组,能够区分1 bp基因编辑突变体与野生型,具有良好的特异性和灵敏度。检测方法可为作物育种筛选节省时间和成本,为基因编辑产品检测监管提供技术支持。

Gene editing technology has been widely used in crop breeding and gene function analysis since its birth.CRISPR/Cas9 system has been the most used gene editing technology at present.For detecting the Os11N3 gene of rice edited by the selfdeveloped CRISPR/Cas9 system,this study designed specific Taqman probes for editing sites,combined with rapid genome extraction technology and mature rice PLD gene primer probes.The specificity of the method was verified by simulating different mutations with synthetic plasmids.The system established in this study could save time and cost while obtaining the genome that met the requirements of qPCR detection.It could distinguish at least 1 bp the gene-edited mutant from the wild type,with good specificity and sensitivity.This system can save time and cost for crop breeding and screening,and provide some technical support for gene editing product testing and supervision.