Single-cell RNA sequencing-based CRISPRi screening resolves molecular drivers of early human endoderm development [set 1]

Studies in vertebrates have outlined conserved molecular control of definitive endoderm (END) development. Yet, recent work also shows that key molecular aspects of human END regulation differ even from rodents. Differentiation of human pluripotent stem cells (hPSCs) to END offers a tractable system to study the molecular basis of normal and defective human-specific END development. Here, we interrogated dynamics in chromatin accessibility during differentiation of hPSCs to END, predicting DNA-binding proteins that may drive this cell fate transition. We then combined single-cell RNA-seq with parallel CRISPR-perturbations to comprehensively define the loss-of-function phenotype of those factors in END development. Following a few candidates, we revealed distinct impairments in the differentiation trajectories for mediators of TGFß signaling and expose a role for the FOXA2 transcription factor in priming human END competence for human foregut and hepatic endoderm specification. Together, this single-cell functional genomics study provides high-resolution insight on human END development. Overall design: CRISPRi screen of endodermal differentiation with a single-cell RNA readout. This submission contains the chromatin data.