Elucidating the epigenetic mechanisms that define the cancer cell-state by exploiting patient-derived organoids
Three-dimensional “organoid” cultures have emerged as a powerful model system to investigate tumor tissues and to capture their inherent heterogeneity. In our laboratory, we have already established several lines of colo-rectal cancer (CRC) derived organoids and performed their morphological, transcriptome and epigenome characterization. We then integrated the generated datasets and used de novo chromatin state discovery to unveil a tumor-specific deregulated enhancerome that is cancer cell-intrinsic thus unveiling a plethora of putative regulatory elements that contribute to shape cancer cell molecular identity.
The proposed PhD project will be dedicated to the implementation of editing tools to modulate the CRC enhancerome and shed light on the relationship of regulatory elements with their target genes. We will employ the nuclease-deficient version (dCas9) of the CRISPR/Cas9 tool, which can be tethered to a diverse array of epigenetic-effector domains for site-specific epigenome modifications. The use of CRISPR/dCas9 epigenetic editing in combination with pooled gRNA libraries and single-cell RNA sequencing technology will allow to understand how specific genome regulatory regions affect the transcriptome at the single-cell level. Ultimately, the identification of key regulatory regions could contribute to dissect the mechanisms underlying cancer cell transcriptional dysregulation and to discover potential targets for epigenetic-based therapeutic approaches.