A pooled single-cell genetic screen identifies regulatory checkpoints in the continuum of the epithelial-to-mesenchymal transition

Integrating single-cell trajectory analysis with pooled genetic screening could reveal the genetic architecture that guides cellular decisions in development and disease. We applied this paradigm to probe the genetic circuitry that controls epithelial-to-mesenchymal transition (EMT). We profiled epithelial cells undergoing a spontaneous, spatially determined EMT in the presence or absence of TGF-β via single-cell RNA-seq. Pseudospatial trajectory analysis identified continuous waves of gene regulation, as opposed to discrete “partial” stages of EMT. KRAS was connected to exit from the epithelial state and acquisition of a fully mesenchymal phenotype. A pooled single-cell CRISPR-Cas9 screen identified EMT-associated receptors and transcription factors, including regulators of KRAS, whose loss impeded progress along EMT. Inhibiting the KRAS effector MEK, and its upstream activators EGFR and MET, demonstrates that interruption of key signaling events reveals regulatory “checkpoints” in the EMT continuum that mimic discrete stages and reconciles opposing views of the program that controls EMT.