Coupled Single-Cell CRISPR Screening and Epigenomic Profiling Reveals Causal Gene Regulatory Networks

Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease. [Display omitted] •A method to measure CRISPR perturbations and chromatin state in single cells•Mapping of dynamic chromatin regulatory networks through intercellular variation•Elucidating principles of epistatic interaction between trans-factors•Perturb-ATAC screen of TF function in epidermal differentiation trajectories Perturb-ATAC combines CRISPR screening with chromatin accessibility profiling of single cells to uncover regulators of chromatin architecture and regulator occupancy and to determine epistatic relationships between regulatory factors in cell fate decisions.