Investigating causative genes for atherosclerosis and coronary heart disease in a zebrafish model system using CRISPR-Cas9technology

Abstract: Coronary artery disease (CAD) is the major cause of death worldwide and atherosclerosis is the major cause of CAD. An unpublished, high-throughput proteomics approach recently identified two of these proteins – Vascular Endothelial Growth Factor A (VEGFA) and Matrix metalloproteinases (MMP12) that were associated with incident CAD in Uppsala-based samples from the prospective investigation of the vascular in Uppsala Seniors (PIVUS) and the Uppsala longitudinal study of adult men studies (ULSAM). We used a zebrafish(Danio rerio) model system to increase our understanding of how these two proteins influence the risk of incident CAD. Zebrafish orthologues of the candidate genes encoding the two proteins of interest (VEGFA and MMP12), and candidate genes that act in the pathways in which VEGFA and MMP12 a reactive, were targeted using a multiplex CRISPR-Cas9 approach. The two multiplexed lines were generated with mmp-13a (mmp13a 202 and mmp13a 204), mmp-13b, mmp-20a, mmp-20b and mmp-30, vefaa, vefab, vegfbb, vegfba, flt1, zfpm2a, zfpm2b, kcnv2a and pum3targeted genes which were injected into the zebrafish carrying transgenic background driven by mpo-mpeg promoter. The founders were crossed and 384 offspring screened per line for whole body lipid and glucose levels, body size and vascular atherosclerosis. Traits were quantified in an automated manner, mutations were mapped using paired-end sequencing, and the data were analysed using zero-inflated negative binomial and multiple linear regression. The present data provided the mechanistic understanding of the targeted genes in developing atherosclerosis. The results showed that the functionally mutated genes have an effect on certain traits such as body size, whole body lipid and glucose, and also on vascular atherogenic traits. The analysis showed significant association of the phenotypes with the mutated genes (p<0.05) which lead to identifying genes which have promising effects on the development of early-stage atherosclerosis. The findings show that mutations in kcnv2a,vegfba, vegfbb, zfpma demonstrates higher risk of vascular inflammation whereas mutation in mmp13a and vegfab shows a decrease in the association with the onset of atherosclerosis and translates into an effect on gene by combining both the orthologues. This provided insight into the role of genes on certain traits thereby influencing the risk of incident CAD.