Somatic Editing of Ldlr With Adeno-Associated Viral-CRISPR Is an Efficient Tool for Atherosclerosis Research

作者：Kelsey E. Jarrett, Ciaran M. Lee, Marco De Giorgi, Ayrea Hurley, Baiba K. Gillard, Alexandria M. Doerfler, Ang Li, Henry J. Pownall, Gang Bao, William R. Lagor · 发表于：Arteriosclerosis Thrombosis and Vascular Biology · 年份：2018 · DOI：10.1161/atvbaha.118.311221 · 被引用次数：79 · 研究领域：CRISPR and Genetic Engineering、Viral Infections and Immunology Research、RNA and protein synthesis mechanisms

Objective- Atherosclerosis studies in Ldlr knockout mice require breeding to homozygosity and congenic status on C57BL6/J background, a process that is both time and resource intensive. We aimed to develop a new method for generating atherosclerosis through somatic deletion of Ldlr in livers of adult mice. Approach and Results- Overexpression of PCSK9 (proprotein convertase subtilisin/kexin type 9) is currently used to study atherosclerosis, which promotes degradation of LDLR (low-density lipoprotein receptor) in the liver. We sought to determine whether CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) could also be used to generate atherosclerosis through genetic disruption of Ldlr in adult mice. We engineered adeno-associated viral (AAV) vectors expressing Staphylococcus aureus Cas9 and a guide RNA targeting the Ldlr gene (AAV-CRISPR). Both male and female mice received either (1) saline, (2) AAV-CRISPR, or (3) AAV-hPCSK9 (human PCSK9)-D374Y. A fourth group of germline Ldlr-KO mice was included for comparison. Mice were placed on a Western diet and followed for 20 weeks to assess plasma lipids, PCSK9 protein levels, atherosclerosis, and editing efficiency. Disruption of Ldlr with AAV-CRISPR was robust, resulting in severe hypercholesterolemia and atherosclerotic lesions in the aorta. AAV-hPCSK9 also produced hypercholesterolemia and atherosclerosis as expected. Notable sexual dimorphism was observed, wherein AAV-CRISPR was superior for Ld...