Recapitulating fibroblast transcriptional states using comprehensive transcription factor perturbations

Cell atlas projects have revealed recurrent transcriptional states across tissues and diseases, but the regulatory mechanisms governing these states remain largely unclear. To investigate whether these states can be recapitulated in vitro, we systematically perturbed 1,836 transcription factors using CRISPRa Perturb-seq in primary fibroblasts and retinal pigment epithelial cells, enabled by a droplet overloading approach that achieves high multiplexing. We observed that CRISPRa activated most transcription factors within a physiological range, that epigenetic features predicted which genes are activatable, and that the protospacer's seed region could drive off-target effects. Perturbations recapitulated gene expression programs resembling in vivo fibroblast states, including universal and inflammatory states, and nominated KLF4 and KLF5 as key regulators of the universal state. Our findings suggest that recurrent transcriptional states reflect gene expression programs that can be elicited via perturbation, opening the door to engineering clinically relevant states for ex vivo study.