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Arterioscler Thromb Vasc Biol. 2018 Sep;38(9):1997-2006. doi: 10.1161/ATVBAHA.118.311221.

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

Author information

1
From the Department of Molecular Physiology and Biophysics (K.E.J., M.D.G., A.H., A.M.D., W.R.L.).
2
Integrative Molecular and Biomedical Sciences Graduate Program (K.E.J.), Baylor College of Medicine, Houston, TX.
3
Department of Bioengineering, Rice University, Houston, TX (C.L., A.L., G.B.).
4
Houston Methodist Research Institute, TX (B.K.G., H.J.P.).
5
Weill Cornell Medicine, New York (B.K.G., H.J.P.).

Abstract

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 Ldlr removal in male mice, while AAV-hPCSK9 was more effective in female mice. Conclusions- This all-in-one AAV-CRISPR vector targeting Ldlr is an effective and versatile tool to model atherosclerosis with a single injection and provides a useful alternative to the use of germline Ldlr-KO mice.

KEYWORDS:

CRISPR-Cas systems; atherosclerosis; gene editing; hypercholesterolemia; lipoproteins

PMID:
30026278
PMCID:
PMC6202188
DOI:
10.1161/ATVBAHA.118.311221
[Indexed for MEDLINE]
Free PMC Article

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