Discovery of Competent Chromatin Regions in Human Embryonic Stem Cells [scRNA-seq]

It remains unknown whether adult-tissue enhancers are pre-established in early embryonic stages. Using CRISPR-activation screens, we found that embryonic stem cells (ESCs) contain transcriptionally competent chromatin regions (CCRs), poised to activate lineage genes before differentiation. CCRs serve as functional enhancer precursors within topological chromatin domains and are pre-marked by pluripotency factors OCT4/NANOG (ON) and DNA demethylation factors QSER1/TET1 (ONQT). We demonstrate that ON and ONQT signatures identify CCRs at a genome-wide scale. CCRs can be harnessed to convert ESCs into differentiated cell types, showcasing their potential for practical applications. Our findings provide insight into how ESCs rapidly establish transcriptional activity during lineage specification, expand our understanding of cellular plasticity and provide the basis to identify adult enhancers in undifferentiated cells. Overall design: Single-cell 10x genomics experiments were performed using hESCs after interrogating candidate CCRs through CRISPRa screens. We employed a gRNA backbone inspired on the CROP-Seq system that allows the dual identification of gRNAs and single-cell transcriptomics. gRNA and transcriptome libraries were generated and sequenced. Final analyses were done using CellRanger and the SCEPTRE algorithm.