One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system

• Published in: Scientific reports 2016-02, Vol.6 (1), p.20620-20620, Article 20620
• Main Authors: Wang, Xianlong, Cao, Chunwei, Huang, Jiaojiao, Yao, Jing, Hai, Tang, Zheng, Qiantao, Wang, Xiao, Zhang, Hongyong, Qin, Guosong, Cheng, Jinbo, Wang, Yanfang, Yuan, Zengqiang, Zhou, Qi, Wang, Hongmei, Zhao, Jianguo
• Format: Article
• Language: English
• Subjects: 45/70; 631/1647/1511; 631/1647/1513/1967/3196; Anatomy; Animal models; Animals; Animals, Genetically Modified - genetics; CRISPR; CRISPR-Cas Systems; Disease Models, Animal; Embryos; Gene loci; Gene Targeting - methods; Genetic Loci; Genomes; Hogs; Humanities and Social Sciences; Humans; Movement disorders; mRNA; multidisciplinary; Neocortex; Neurodegenerative diseases; PARK7 protein; Parkin protein; Parkinson Disease - genetics; Parkinson Disease - metabolism; Parkinson's disease; Protein Deglycase DJ-1 - genetics; Protein Deglycase DJ-1 - metabolism; Protein Kinases - genetics; Protein Kinases - metabolism; PTEN-induced putative kinase; Science; Swine; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep20620

Pig shows multiple superior characteristics in anatomy, physiology, and genome that have made this species to be more suitable models for human diseases, especially for neurodegenerative diseases, because they have similar cerebral convolutions compared with human neocortex. Recently, CRISPR/Cas9 system shows enormous potential for engineering the pig genome. In this study, we expect to generate human Parkinson’s disease pig model using CRISPR/Cas9 system by simultaneously targeting three distinct genomic loci, parkin/DJ-1/ PINK1 , in Bama miniature pigs. By co-injection of Cas9 mRNA and multiplexing single guide RNAs (sgRNAs) targeting parkin, DJ-1 , and PINK1 genes, respectively, into in vivo derived pronuclear embryos, we simultaneously targeted three distinct genomic loci. The gene modified piglets remain healthy and display normal behavior at the age of 10 months. In addition, despite the high number of sgRNAs were employed in the present study, our trio-based whole-genome sequencing analysis suggested that the incidence of off-target events is low. Our results demonstrate that the simplicity, efficiency, and power of the CRISPR/Cas9 system to allow for the modification of multiple genes in pigs and yield results of high medical value.