Generation Of ApoE^-/- Pigs By CRISPR/Cas9 Technology

Background:Cardiovascular and cerebrovascular diseases, a leading cause of mortality worldwide, are caused mainly by atherosclerosis, a chronic inflammatory disease of the arterial wall. The underlying pathogenesis of atherosclerosis remains elusive, however, hyperlipidemia and hypercholesterolemia are considered as the most important risk factors of atherosclerosis. Apolipoprotein E (ApoE) plays an important role in cholesterol and lipoprotein transport and metabolism. Although ApoE knockout mice have been used as model of atherosclerosis and led to an understanding the mechanisms of atherosclerosis development, it is not a suitable model for human atherosclerosis development. Production of genetically engineered pig models has several advantages over rodent models due to its similar coronary anatomy and physiology to humans.Objective:In this study, highly efficient CRISPR/Cas9 system will be used to target and disrupt ApoE gene in pigs to produce the first ApoE-/- pigs in the world. The ApoE-/- pigs could be useful models for providing further insight into pathogenesis of atherosclerosis and clues to assess novel prevention and treatment applications.Methods:CRISPR/Cas9 targeting plasmid (Cas9-sgRNA) were designed and constructed based on the genomic sequence of porcine ApoE. Cas9 plasmid were then co-transfected with a neomycin-expression plasmid (pCMV-tdTomato) into porcine fetal fibroblasts(PFFs) by nucleofection method. After G418 selection,cell colonies with biallelic mutation of ApoE gene were identified by PCR sequencing. With the aid of somatic cell nuclear transfer technology, cell pools with biallelic mutation were used as nuclear donors to produce cloned ApoE gene mutant piglets.For cloned piglets, PCR sequencing was used to determine their genotypes.Results:Cas9-sgRNA plasmid was designed and constructed.Then we transfected the plasmid into PFFs and screened cell colonies with ApoE gene mutations.14 and 27 ApoE biallelic modified colonies were obtained from PFFs of Zhonghua Miniature pigs and Bama suckling pigs,respectively.By applying the SCNT technology,17 cloned piglets were produced.The genotypes of the cloned piglets were all confirmed as ApoE gene mutant and the mutation patterns were in accord with the donor cells.Conclusion:Our study indicates that the CRISPR/Cas9 system combined with SCNT technology is an efficient approach for one-step generating ApoE-/- pigs. And the ApoE-/- pigs as atherosclerosis models could provide further insight into pathogenesis of atherosclerosis and assess novel prevention and treatment applications.