CRISPR-Cas9 Screens Reveal Genes Regulating a G0-like State in Human Neural Progenitors

The coordination of developmental potential and proliferation in stem and progenitor cells is essential for mammalian development and tissue homeostasis. We performed CRISPR-Cas9 screens in human neural progenitor cells (hNPCs) and identified genes, including CREBBP, NF2, PTPN14, TAOK1, or TP53, that limit expansion. Knockout of these genes causes increased hNPC proliferation via skipping of a transient G0-like state, characterized by expression of genes associated with quiescent neural stem cells and neural development and molecular features of quiescent cells (e.g., hypophosphorylated Rb, low CDK2 activity, and p27 stabilization). Single-cell RNA- sequencing of hNPCs revealed distinct G0/G1 populations, altered in G0-skip mutants through both distinct and convergent downstream effectors, including cell cycle, Hippo- YAP, and novel targets. Our results provide a molecular and phenotypic portrait of expanding hNPCs including a gene expression map of their cell cycle and characterization of antiproliferative factors that regulate cell cycle exit with likely roles in maintaining developmental potential.