A single-cell CRISPR screen defines a gene regulatory network governing human pluripotency in primed and naive cells

Human pluripotent stem cells (hPSCs) can self-renew indefinitely and differentiate into all three embryonic germ layers. We previously defined the essentialome of hPSCs using a genome-wide CRISPR screen, but the functions of each gene remain obscure. Here, we used a pooled single-cell CRISPR screen to investigate pluripotent-specific essential transcription factors (TFs). We found that most TFs form a highly interconnected gene regulatory network (GRN) that governs key aspects of pluripotency, including self-renewal, differentiation, survival, and transposable element expression. Interestingly, we identify multiple TFs that act as lineage-specific gatekeepers, blocking exit from pluripotency, and others that inhibit pluripotency, potentially balancing self-renewal and differentiation responsiveness. Finally, perturbing the GRN in naive hPSCs revealed both conserved and state-specific regulatory roles relative to primed cells. Altogether, our analysis defines an extended GRN for human pluripotency, offering insights into early human development. These findings may inform strategies to improve hPSC-based disease models and regenerative therapies.