Enhancer-gene regulatory interactions in colorectal cancer revealed through genome-wide CRISPRi perturbations

<ns5:p>Background Establishing 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. Results We 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 &lt; 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. Conclusions We 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.</ns5:p>