Rationale Individuals with naturally occurring loss-of-function PCSK9 mutations experience reduced blood low-density lipoprotein cholesterol (LDL-C) levels and protection against cardiovascular disease. Objective The goal of this study was to assess whether genome editing using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system can efficiently introduce loss-of-function mutations into the endogenous PCSK9 gene in vivo. Methods and Results We used adenovirus to express Cas9 and a CRISPR guide RNA targeting Pcsk9 in mouse liver, where the gene is specifically expressed. We found that within three to four days of administration of the virus, the mutagenesis rate of Pcsk9 in the liver was as high as >50%. This resulted in decreased plasma PCSK9 levels, increased hepatic LDL receptor levels, and decreased plasma cholesterol levels (by 35%–40%) in the blood. No off-target mutagenesis was detected in 10 selected sites. Conclusions Genome editing with the CRISPR-Cas9 system disrupts the Pcsk9 gene in vivo with high efficiency and reduces blood cholesterol levels in mice. This approach may have therapeutic potential for the prevention of cardiovascular disease in humans.

Rationale Non-coding gene variants at the SORT1 locus are strongly associated with LDL-C levels as well as with coronary artery disease (CAD). SORT1 encodes a protein called sortilin, and hepatic sortilin modulates LDL metabolism by targeting apoB-containing lipoproteins to the lysosome. Sortilin is also expressed in macrophages, but its role in macrophage uptake of LDL and in atherosclerosis independent of plasma LDL-C levels is unknown. Objective To determine the effect of macrophage sortilin expression on LDL uptake, foam cell formation, and atherosclerosis. Methods and Results We crossed Sort1−/− mice onto a ‘humanized’ Apobec1−/−; hAPOB Tg background and determined that Sort1 deficiency on this background had no effect on plasma LDL-C levels but dramatically reduced atherosclerosis in the aorta and aortic root. In order to test whether this effect was a result of macrophage sortilin deficiency, we transplanted Sort1−/−;LDLR−/− or Sort1+/+;LDLR−/− bone marrow into Ldlr−/− mice and observed a similar reduction in atherosclerosis in mice lacking hematopoetic sortilin without an effect on plasma LDL-C levels. In an effort to determine the mechanism by which hematopoetic sortilin deficiency reduced atherosclerosis, we found no effect of sortilin deficiency on macrophage recruitment or LPS-induced cytokine release in vivo. In contrast, sortilin deficient macrophages had significantly reduced uptake of native LDL ex vivo and reduced foam cell formation in vivo, whereas sortilin overexpression in macrophages resulted in increased LDL uptake and foam cell formation. Conclusions Macrophage sortilin deficiency protects against atherosclerosis by reducing macrophage uptake of LDL. Sortilin-mediated uptake of native LDL into macrophages may be an important mechanism of foam cell formation and contributor to atherosclerosis development.