Fig. 6. Experimental interrogation of a new CAD locus near MYO9B.

a, Regional association plot from the primary CAD meta-analysis for the new gene-dense region around MYO9B. Colored dots represent the position (x axis) in GRCh37 coordinates and –log₁₀(meta-analysis P value) (y axis) of each variant in the region. Dots are shaded to represent the r² with the lead CAD variant (rs7246865), estimated using a random sample of 5,000 European ancestry participants from the UK Biobank. Recombination peaks are plotted in blue based on estimates of recombination from 1000 Genomes European ancestry individuals. b, Identification of a noncoding enhancer in the region around the CAD association signal. The plot shows an inset of a 5-kb window surrounding the lead CAD variant (rs7246865). The top three tracks (blue) show H3K27Ac ChIP-seq of human CA, aorta and tibial artery, identifying a vascular tissue enhancer element overlying rs7246865. The bottom three tracks (purple) show ATAC-seq of human monocytes, immortalized human aortic ECs and CA-VSMCs, identifying a region of open chromatin in all three cell types around rs7246865. The plot also shows the location of the sgRNAs used for deletion of the noncoding enhancer. c, Efficiency of CRISPR editing in primary human cells. The Cas9-sgRNA ribonucleoprotein nucleofection method resulted in noncoding enhancer deletion efficiency (x-axis) of greater than 0.5 by densitometry and was comparable across monocytes, ECs and CA-VSMCs. Points indicate enhancer deletion efficiency for each of the 12 replicates. Horizontal bars indicate mean enhancer deletion efficiency, and whiskers indicate 95% CIs. d, Relative expression of nearby genes after enhancer deletion in ECs. The y axis shows mean expression of five local genes expressed in ECs compared to expression levels of a control gene (GAPDH). Blue bars indicate gene expression with Cas9–control sgRNA. Red bars indicate expression with tandem enhancer-deleting guides as identified in b. Points indicate gene expression levels for each of the six replicates. Vertical bars indicate mean expression levels and whiskers indicate 95% CIs. Gene expression was quantified by qPCR. Expression levels were compared using an unpaired two-way Student’s t test. Reduced expression of MYO9B and HAUS8 was identified after 131-bp enhancer deletion as in b. **P = 0.0020; ***P < 0.0001. e, Relative expression of nearby genes after enhancer deletion in CA-VSMCs. The y-axis shows mean expression of five local genes expressed in CA-VSMCs compared to expression levels of a control gene (GAPDH). Blue bars indicate gene expression with Cas9–control sgRNA. Red bars indicate expression with tandem enhancer-deleting guides as identified in b. Points indicate gene expression levels for each of the six biological replicates. Vertical bars indicate mean expression levels and whiskers indicate 95% CIs. Gene expression was quantified by qPCR. Expression levels were compared using an unpaired two-way Student’s t test. Reduced expression of MYO9B was identified after 131-bp enhancer deletion as in b. **P = 0.0044. f, In vitro endothelial wound healing with enhancer and gene deletions. The y-axis indicates fluorescence intensity, a read-out for endothelial wound healing and a composite of migration and proliferation. ECs with CRISPR–Cas9 genome editing for enhancer deletion (red) or single-gene knock-outs exhibited diminished wound healing relative to nontargeting control with no deletions (blue). Dots indicate endothelial wound healing for each of the six replicates. Vertical bars indicate mean wound-healing levels and whiskers indicate 95% CIs. Levels of wound healing were compared by one-way ANOVA. *P = 0.0464; **P = 0.0013; ***P = 0.0003; ****P < 0.0001; NS, not significant.