基于CRISPR/Cas9系统的多基因敲除载体的构建及其敲除效率检测  

Construction and Knockout Efficiency Detection of Multiple Knockout Vector Based on the CRISPR/Cas9 System

作　　者：徐磊[1] 赵育蓉 胡悦旻 王昊 彭玥晗 鞠辉明[1,2,3] XU Lei;ZHAO Yu-Rong;HU Yue-Min;WANG Hao;PENG Yue-Han;JU Hui-Ming(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Collaborative Innovation Center for Animal Epidemics and Zoonoses,Yangzhou 225009,China;College of Guangling,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院,扬州225009 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009 [3]扬州大学广陵学院,扬州225009

出　　处：《农业生物技术学报》2022年第5期1023-1030,共8页Journal of Agricultural Biotechnology

基　　金：江苏省自然科学基金(BK20201224);国家自然科学基金(31872323);江苏高校优势学科建设工程资助项目(2018);江苏高校品牌专业建设工程资助项目(TAAP,2018-2022);扬州大学大学生创新创业训练计划项目(X20200705)。

摘　　要：CRISPR/Cas9系统是近年来快速发展的一种简单高效的基因编辑系统。在哺乳动物中,同时靶向敲除多基因的研究仍然较为匮乏。为优化哺乳动物中靶向多基因的敲除系统的构建,本研究在已有的CRISPR/Cas9载体的基础上进行升级改造,将4个U6启动子介导的小向导RNA(small guide RNA,sgRNA)表达框串联至一个载体中的形式制备了多基因位点编辑载体,分别选取家猪(Susscrofa)的去乙酰化酶3基因(sirtuin 3,Sirt3)和围脂滴蛋白1基因(perilipin 1,Plin1)的2个sgRNA,构建了2个猪Sirt3 sgRNA的基因编辑载体(S2)、2个猪Plin1 sgRNA基因编辑载体(P2)和同时包括上述4个sgRNA的基因编辑载体(S2P2),上述载体分别转染猪肾细胞PK15,以转染未装载靶点质粒的细胞为对照组(CON组),在各实验组细胞目的基因的DNA水平、RNA水平和蛋白水平检测目的基因敲除效率。结果表明,各实验组细胞目的基因DNA中均检测出突变,S2P2组和S2组中Sirt3基因突变率分别为33%和26%,S2P2组和P2组中Plin1基因突变率分别为33%和24%;对目的基因RNA水平与蛋白水平的检测结果表明,与CON组相比,所有实验组的目标基因mRNA及蛋白表达量均有所下降,差异极显著(P<0.01),其中S2组与S2P2组间Sirt3基因表达量差异不显著;P2组与S2P2组间Plin1基因表达量差异不显著。本研究通过改良的CRISPR/Cas9载体系统构建了可以同时对猪Sirt3基因和Plin1基因高效编辑的载体,有助于后续开展多基因功能的研究。The CRISPR/Cas9 system is a simple and efficient gene editing system that has been rapidly developed in recent years.In mammals,research on knocking out multiple genes simultaneously is still scarce.In order to optimize the construction of a knock-out system targeting multiple genes in mammals,the existing CRISPR/Cas9 vector was upgraded in this study,a multi-targets knock-out plasmid based on CRISPR/Cas9 system cascading by 4 small guide RNA(sgRNA)expression cassettes mediated by U6 promoters was constructed.Sirtuin 3 gene(Sirt3)and Perilipin 1 gene(Plin1)of Sus scrofa were chosen to assess the upgraded plasmid.2 sgRNA targeting Plin1 and Sirt3 were selected respectively,and the knock-out vectors were constructed by inserting the sgRNA based on the upgraded plasmid.In this study,PK15 were divided into 4 groups.The control group CON was the cells transfected by the upgraded plasmid without any targets.The group S2 was the cells cotransfected 2 vectors targeting the Sirt3,while the group P2 was cotransfected 2 vectors targeting the Plin1,and the group S2P2 was cotransfected the vector contains all the targets.Detect the target gene mutation rate of each experimental group cell at the cell genomic DNA level,and use qPCR and Western blot to determine expression of RNA and protein of the target gene expression of each group of cells.The results showed that mutations were detected in the cells of each experimental group.Mutation rates of Sirt3 gene in S2P2 and S2 were 33%and 26%,respectively.Plin1 gene mutation rates in S2P2 and P2 groups were 33%and 24%,respectively.And compared with the CON group,the target mRNA and protein expression levels decreased in all experimental groups,the difference was extremely significant(P<0.01).Among them,there was no significant difference in Sirt3 gene expression between S2 group and S2P2 group and in Plin1 gene expression between P2 group and S2P2 group.In this study,a vector that can simultaneously knockout porcine Sirt3 and Plin1 genes was constructed through the improved CRISPR/

关 键 词：CRIPSR/Cas9 多基因敲除 载体构建 

分 类 号：S828.8[农业科学—畜牧学] Q812[农业科学—畜牧兽医]