title: One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system.
abstract: 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.
authors: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Xianlong
authors: X
authors: Wang
authors: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Chunwei
authors: C
authors: Cao
authors: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Jiaojiao
authors: J
authors: Huang
authors: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Jing
authors: J
authors: Yao
authors: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Tang
authors: T
authors: Hai
authors: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Qiantao
authors: Q
authors: Zheng
authors: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Xiao
authors: X
authors: Wang
authors: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Hongyong
authors: H
authors: Zhang
authors: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Guosong
authors: G
authors: Qin
authors: State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors: Jinbo
authors: J
authors: Cheng
authors: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
authors: Yanfang
authors: Y
authors: Wang
authors: State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors: Zengqiang
authors: Z
authors: Yuan
authors: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Qi
authors: Q
authors: Zhou
authors: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Hongmei
authors: H
authors: Wang
authors: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Jianguo
authors: J
authors: Zhao
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D064113:CRISPR-Cas Systems; D004195:Disease Models, Animal; D018390:Gene Targeting / Q000379:methods*; D056426:Genetic Loci; D006801:Humans; D010300:Parkinson Disease / Q000235:genetics* / Q000378:metabolism; D000071617:Protein Deglycase DJ-1 / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D013552:Swine; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D000099202:PTEN-Induced Putative Kinase
chemical_list: D000071617:Protein Deglycase DJ-1; D011494:Protein Kinases; D044767:Ubiquitin-Protein Ligases; D000099202:PTEN-Induced Putative Kinase; C111567:parkin protein