Abstract 15608: Validation of Enhancer/Silencer Activity of the Intracranial Aneurysm-Associated SNP at SOX17 in Primary Human Endothelia

Abstract

Introduction: GWAS have identified multiple SNPs that occur more commonly in those with intracranial aneurysms. Many SNPs are located in noncoding genomic regions, suggesting that genetic risk may operate on functional regulatory elements that influence gene expression. However, regulatory elements are most often only indirectly identified through methods such as DNAse hypersensitivity without direct assessment of activity. Hypothesis: We hypothesize CRISPR mediated ablation of putative regulatory elements located within intergenic loci associated with IA will result in mRNA transcript modulation of genes located within the respective topology associated domain. Methods: We investigated a SNP within a haplotype block near SOX17. The haplotype was predicted to have active enhance activity, and to mediate SOX17 expression, based on assessment of its topologically associated domain. Lentiviral constructs (each with unique antibiotic resistance and fluorescent protein reporters) for the expression of Cas9 nuclease and two gRNAs flanking a putative element located at chr8:54412191-54413910 were transfected into the Lenti-X viral propagating cell line (Figure, left). Viral supernatants were transduced into primary HUVEC cells by spinfection with 5 ug/mL polybrene. After antibiotic selection and fluorescence validation, mRNA was isolated, and transcripts quantified through PCR. Results: Lentiviral transduction of HUVECs resulted in CRISPR-mediated removal of the enhancer element located within the haplotype block chr8:54397171-54415556 and resulted in a substantial increase of the SOX17 gene mRNA located within its topology associated domain (Figure, right). Conclusions: CRISPR mediated ablation was used to efficiently remove and validate a genomic element with putative regulatory activity in HUVECs. The methods will be employed to study additional regulatory elements with IA associations as well as other primary vascular cells.