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

Human pluripotent stem cells (hPSCs) can give rise to the three embryonic germ layers and possess a limitless propagation potential. We have previously defined the essentialome of hPSCs through a genome-wide CRISPR screen, but the function of each essential gene is still obscure. Here, we uncovered the role of pluripotent-specific transcription factors (TFs) that are essential in hPSCs, using a pooled CRISPR screen combined with single-cell transcriptomics. We show that most of these TFs form a remarkably interconnected gene regulatory network (GRN), controlling multiple aspects of pluripotency, spanning regulation of differentiation, self-renewal, cell survival, and expression of transposable elements. Interestingly, various TFs function as positive regulators of pluripotency, serving as gatekeepers from differentiation, and preventing transition into different cell lineages in a TF-specific manner. Surprisingly, other TFs inhibit the pluripotency network, potentially balancing its maintenance with responsiveness to differentiation cues, which we could validate through individual gene knockouts. Finally, by perturbing the TFs network in naïve cells, we reveal both shared and distinct roles for the GRN between the two pluripotency states. In summary, this analysis enabled the construction of an extended network for human pluripotency. This study sheds new light on principles in early human development and can hopefully be used to enhance the use of hPSCs for disease modelling or cell therapy.