Construction and Knockout Efficiency Detection of Multiple Knockout Vector Based on the CRISPR/Cas9 System
XU Lei1, ZHAO Yu-Rong1, HU Yue-Min1, WANG Hao1, PENG Yue-Han1, JU Hui-Ming1,2,3,*,
1 College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China; 2 Jiangsu Collaborative Innovation Center for Animal Epidemics and Zoonoses, Yangzhou 225009, China; 3 College of Guangling, Yangzhou University, Yangzhou 225009, China
Abstract：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/Cas9 vector system. This research is helpful to the subsequent study of multi-gene function.
徐磊, 赵育蓉, 胡悦旻, 王昊, 彭玥晗, 鞠辉明. 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[J]. 农业生物技术学报, 2022, 30(5): 1023-1030.
XU Lei, ZHAO Yu-Rong, HU Yue-Min, WANG Hao, PENG Yue-Han, JU Hui-Ming,. Construction and Knockout Efficiency Detection of Multiple Knockout Vector Based on the CRISPR/Cas9 System. 农业生物技术学报, 2022, 30(5): 1023-1030.
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