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

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.

Comparison of CRISPR/Cas endonucleases forin vivoretinal gene editing

“…NmeCas9 (Ibraheim et al, 2018) to a variety of somatic tissue in mice (Ran et al, 2015;Ibraheim et al, 2018;Jarrett et al, 2018;Pan et al, 2018;Xu et al, 2019) . Despite the encouraging in vivo application of these CRISPR/Cas systems, delivered via dual or all-in-one vectors, it is not clear which are the most efficacious for retinal editing in vivo .…”

PCSK9 Variants in Familial Hypercholesterolemia: A Comprehensive Synopsis

“…Although viral vectors have higher delivery efficiency and adeno-associated viral vectors have recognized efficiency in atherosclerosis research, their biosecurity issues hinder their wide application. Based on the nanocarrier-delivered CRISPR/Cas9 system, Zhang et al (2019) use a triple targeting strategy to produce a LOF variant in the PCSK9 gene and this strategy might be a potential target therapy for CVD without side effects ( Jarrett et al, 2018 ). In addition, precise knock-in of specific nucleotide changes have proven to be inefficient in non-proliferating cells in vivo .…”

Delivery Approaches for Therapeutic Genome Editing and Challenges

“…The CRISPR-Cas9 from Staphylococcus aureus (SaCas9) uses an sgRNA to bind to sites upstream of a 5′-NNGRRT PAM sequence. The SaCas9 has been employed in preclinical studies using animal models of human diseases that demonstrated successful therapeutic application [ 55 , 56 ]. In addition, SaCas9 reagents, similar to other Cas homologs, are commercially available to enable ease of their direct delivery as preformed sgRNA-Cas9 RNPs for potent cleavage activity at endogenous sites in cells.…”