Project Summary/Abstract Early mammalian embryogenesis is characterized by robust cellular proliferation, maintenance of pluripotency, and resistance to differentiation until gastrulation, when cells must commit to specific lineages. Pluripotent stem cells in the early mammalian embryo progress through developmental states in preparation for lineage specification during gastrulation, but how pluripotent stem cells become competent to differentiate is not well understood. Two distinct states of pluripotency can be replicated in vitro by using mouse embryonic stem cells (ES) for a naïve state and epiblast like cells (EpiLC) for a primed state. The naïve state and primed state cells recapitulate pluripotent cells that are resistant to differentiation and competent to differentiation, respectively. Naïve ES cells can rapidly transition to the EpiLC, primed state with simple changes to cell culture media. This transition is irreversible, so EpiLC die if they are returned to naïve cell culture conditions. Previously, several genes have been shown to be necessary for the transition from naïve to primed states, and inactivation of these genes prevents cell death when cells are switched between EpiLC and ES culture conditions. To screen for novel genes required for exit out of naïve pluripotency, I performed a CRISPR-cas9 genome-wide pooled knockout screen and targeted all protein coding genes in the mouse genome with ~90k unique sgRNAs. The screen yielded 40 high confidence candidates (FDR<10%), including genes known to be required for naïve to primed transition, such as: Tcf7L1, Zfp281, Tsc1, Tsc2, and Flcn. Novel genes were also identified; interestingly, many of these genes affect endocytic trafficking to the lysosome. There was a particularly strong enrichment for genes that are part for the HOPS complex important for late endolysosome fusion and mTOR pathway genes involved in amino acid sensing on lysosomes. This proposal aims to confirm my screen findings and elucidate roles for late-endosome-lysosome fusion and mTOR pathway/autophagy in exit from the naïve state. Demonstrating these roles will provide significant new insight into how cytoplasmic processes function to enable pluripotent cells to become competent to differentiate. Therefore, the findings of this proposal will be critical to understanding both basic developmental biology as well as deriving application from pluripotent stem cells for regenerative medicine and cell-based therapies.