Liver-directed genome editing for the study and correction of lipid disorders

Atherosclerotic Vascular Disease (AVD) is the major underlying cause of both heart attack and stroke. Given that current methods to query the impact of a single gene on lesion development require years of mouse breeding to achieve homozygous double knockout with Ldlr on a C57BL/6 background, more rapid methods are required to investigate the ever-growing list of candidate genes being identified through human genetics. The Clustered Regularly-Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) system is a gene-editing tool that mediates very precise genetic disruptions. By packaging the CRISPR/Cas9 system into Adeno Associated Viral (AAV) vectors, we aimed to achieve targeted delivery and efficient gene editing in adult mice. We hypothesized that somatic disruption of Ldlr using AAV-CRISPR could generate atherosclerosis in adult mice. Adeno-Associated Viral (AAV) vectors based on serotype 8 were used to deliver small guide RNAs (gRNA) to adult Cas9 transgenic mice. The mice received 1) a nontargeting gRNA, 2) a gRNA targeting Ldlr, or 3) gRNAs to both Ldlr and Apob. Mice were placed on a Western diet and followed for twenty weeks for changes in plasma cholesterol and susceptibility to atherosclerosis. Disruption of Ldlr with AAV-CRISPR was robust, resulting in severe hypercholesterolemia (Ldlr: 728 +/- 174 mg/dl vs. control: 350 +/- 18.7 mg/dl) and atherosclerotic lesions in the aorta (Ldlr: 2.21% +/- 2.10% vs. control: 0.0% lesion area) when maintained on a western diet. Mice receiving gRNAs to both Ldlr and Apob had an identical degree of Ldlr disruption, but dramatically lower cholesterol (125 +/- 27.3 mg/dl), profound hepatic steatosis, and a complete prevention of western-diet induced atherosclerosis. These data show that the AAV-CRISPR system is a valuable, and immediately useful, time-saving tool for atherosclerosis studies that can also be used to test the involvement of liver-expressed candidate genes in this disease.