In post GWAS studies, two main problems emerge: identifying the correct susceptibility gene(s) for each locus and their functional role in disease pathogenesis and identifying the causal genetic variant(s) which drive disease development.
Common variants that influence the activity of specific regulatory elements such as enhancers, may affect target gene expression through direct, physical interactions. How do we overcome linking candidate risk variants that occur at a distance from putative target genes?
By using a two-pronged approach, 3C/4C technologies can be utilized to help identify physical interactions between risk variants and target genes. Second, we established specific enhancer profiles by epigenetic profiling all ovarian cancer histologic subtypes. Enhancer marks that overlap with positive 3C/4C SNP interactions are later validated in in-vitro neoplastic models of ovarian cancer.
Circular Chromosome Conformation Capture (4C)
4C is an unbiased, robust, high resolution assay to evaluate the interactions between a region of interest and the rest of the genome (a one-to-all assay).
4C analysis is used to identify cis-interacting regions that are shared between breast and ovarian cancer cell lines.
Once candidate ovarian cancer susceptibility genes are physically linked to specific variant, these positive interactions are then prioritized for further characterization in CRISPR genome editing assays.
CRISPR-Cas9 Gene Editing
CRISPR-Cas9 based technologies have given us the ability to confirm the function of these plausible functional variants in regulatory regions.
The fusion of deactivated Cas9 (dCas9) to gene-regulatory proteins, inhibitory CRISPRi and activation CRISPRa, has given researchers the ability to functionally probe non-coding genomic regions for transcriptional downregulation and upregulation activity. Using CRISPRi, chromatin looping regulatory elements (4C interacting regions) can be perturbed and downregulated to investigate the transcriptional output of their target gene.