Understanding the effects of genetic perturbations on the cellular state has been challenging using traditional pooled screens, which typically rely on the delivery of a single perturbation per cell and unidimensional phenotypic readouts. Here, we use barcoded open reading frame overexpression libraries coupled with single-cell RNA sequencing to assay cell state and fitness, a technique we call SEUSS (scalable functional screening by sequencing). Using SEUSS, we perturbed hPSCs with a library of developmentally critical transcription factors (TFs) and assayed the impact of TF overexpression on fitness and transcriptomic states. We further leveraged the versatility of the ORF library approach to assay mutant genes and whole gene families. From the transcriptomic responses, we built genetic co-regulatory networks to identify altered gene modules and found that KLF4 and SNAI2 drive opposing effects along the epithelial-mesenchymal transition axis. From the fitness responses, we identified ETV2 as a driver of reprogramming toward an endothelial-like state
