CRISPR/Cas9-mediated <i>ApoE</i>-/- and <i>LDLR</i>-/- double gene knockout in pigs elevates serum LDL-C and TC levels

Abstract

// Lei Huang 1, 3 , Zaidong Hua 2 , Hongwei Xiao 2 , Ying Cheng 1 , Kui Xu 1 , Qian Gao 1 , Ying Xia 1 , Yang Liu 1 , Xue Zhang 1 , Xinming Zheng 2 , Yulian Mu 1 and Kui Li 1 1 State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China 2 Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Science and Veterinary Medicine, Hubei Academy of Agricultural Science, Wuhan 430064, China 3 Animal Functional Genomics Group, Agricultural Genomes Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China Correspondence to: Yulian Mu, email: mouyulian@caas.cn Keywords: cardiovascular disease, dyslipidemia, animal model, multiple-gene knockout, gene editing Received: September 06, 2016&nbsp;&nbsp;&nbsp;&nbsp; Accepted: March 28, 2017&nbsp;&nbsp;&nbsp;&nbsp; Published: April 17, 2017 ABSTRACT The traditional method to establish a cardiovascular disease model induced by high fat and high cholesterol diets is time consuming and laborious and may not be appropriate in all circumstances. A suitable pig model to study metabolic disorders and subsequent atherosclerosis is not currently available. For this purpose, we applied the CRISPR/Cas9 system to Bama minipigs, targeting apolipoprotein E (ApoE) and low density lipoprotein receptor (LDLR) gene simultaneously. Six biallelic knockout pigs of these two genes were obtained successfully in a single step. No off-target incidents or mosaic mutations were detected by an unbiased analysis. Serum biochemical analyses of gene-modified piglets showed that the levels of low density lipoprotein choleserol (LDL-C), total cholesterol (TC) and apolipoprotein B (APOB) were elevated significantly. This model should prove valuable for the study of human cardiovascular disease and related translational research.