Enhancer-gene regulatory interactions in colorectal cancer revealed through genome-wide CRISPRi perturbations
BackgroundEstablishing functional relationships between distal enhancers and their target genes is a central challenge in genome biology and cancer genetics. CRISPR/dCas9-mediated perturbations followed by single-cell RNA sequencing (Perturb-seq) has emerged as an efficient means of establishing enhancer-target gene relationships. ResultsWe used genome-wide CRISPRi Perturb-seq with 35,139 guide RNAs targeting 12,117 enhancers to create a functional enhancer-gene map of colorectal cancer (CRC), identifying 238 significant regulatory associations (FDR < 0.1). Integration of chromatin accessibility (ATAC-seq), histone modification profiling (ChIP-seq), and ultra-high resolution chromatin conformation capture (Micro-C) data revealed that enhancer regulation in CRC is constrained by topological domains and often targets the nearest gene, findings corroborated by Activity-by-Contact modelling. ConclusionsWe present here a comprehensive, genome-scale functional atlas of enhancer-gene associations in CRC. This resource should provide a foundation for studying gene regulation in colorectal tumorigenesis and for prioritising candidate non-coding drivers in cancer sequencing studies. PLAIN LANGUAGE SUMMARYO_ST_ABSMapping the hidden DNA switches that control colon cancer genesC_ST_ABSThe total amount of DNA in humans is vast, but only a tiny proportion of it is directly related to genes. While people may know that genes act as blueprints for the body, there are also short stretches of DNA called "enhancers", that fine-tune how strongly genes are activated. When these enhancers get damaged or mutated, it can trigger disease, suggesting that changes to these "control switches" themselves, and not just the genes, can be a driver of cancer. Thus, understanding how these enhancers function is key to uncovering the mechanisms that drive colorectal cancer. In this study, we aimed to map the role of enhancers in colorectal (bowel) cancer development. We used Perturb-seq, a cutting-edge genetic tool that allowed us to systematically turn off more than 12,000 individual enhancers, to identify which ones control which genes. We successfully linked 238 enhancers to the genes they regulate. Combining these results with other biological information, such as the 3D-shape of the DNA, allowed us to build a detailed picture of how enhancers physically interact with their target genes inside cancer cells. This work provides a comprehensive "wiring diagram" of enhancer-gene connections in colorectal cancer, and a resource for researchers seeking to develop new treatment approaches.