Mapping dynamic transcription factor hierarchies using pooled CRISPR screening with single-cell epigenome readout

Cellular state transitions, such as those occurring during cancer progression, involve transcriptional switches that are controlled by specific combinations of transcription factors. How such dynamic processes are encoded in the genome sequence as “regulatory programs”, and how transcription factors control the epigenome and activate specific enhancers, remains largely unknown. Single-cell transcriptomics provide a unique opportunity to unravel these regulatory programs during cellular state transitions. Here, we propose to exploit CRISPR-based screening to perturb hundreds of transcription factors and chromatin modifiers in parallel, followed by combined single-cell transcriptome and single-cell epigenome readouts. To achieve this, we will develop combined single-cell RNA+ATAC approaches using droplet microfluidics. We will apply this single-cell perturbation approach to a collection of in-house established cancer state transitions, whereby cancer cells transition to mesenchymal-like drug-resistant cell states. Once established, we will also apply this technique to 3D tumor-on-chip models, and to primary cells during differentiation. Combined with advanced computational analyses, this systems biology project will provide unprecedented resolution to map genomic regulatory programs.