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Genetic Dissection of the Exit of Pluripotency in Mouse Embryonic Stem cells by CRISPR-based Screening 10.17863/CAM.24867 Li , Meng Yusa , Kosuke The ground state naive pluripotency is established in the epiblast of the blastocyst and can be captured by culturing mouse embryonic stem cells (mESCs) with MEK and GSK3 inhibitors (2i). The transcription network that maintains pluripotency has been extensively studied with the indispensable core factors being Oct4, Sox2 and Nanog, together with other ancillary factors reinforcing the network. However, how this network is dissolved at the onset of differentiation is still not fully understood. To identify genes required for differentiation in an unbiased fashion, I conducted a genome-wide CRISPR-Cas9-mediated screen in Rex1GFPd2 mESCs. This cell line expresses GFP specifically in the naive state and rapidly down-regulate upon differentiation. I differentiated mutagenised mESCs for two days and sorted mutants that kept higher GFP expression. gRNA representation was subsequently analysed by sequencing. I identified 563 and 8 genes whose mutants showed delayed and accelerated differentiation, respectively, at a false discovery rate (FDR) cutoff of 10%. The majority of the previously known genes were identified in my screen, suggesting faithful representation of genes regulating differentiation. Detailed screening result analysis revealed a comprehensive picture of pathways involved in the dissolution of naive pluripotency. Amongst the genes identified are 19 mTORC1 regulators and components of the mTORC2 complex. Deficiency in the TSC and GATOR complexes resulted in mTORC1 upregulation in consistent with previous studies. However, they showed opposite phenotype during ESC differentiation: TSC complex knockout cells showed delayed differentiation, whereas GATOR1 deficiency accelerated differentiation I found that the pattern of GSK3b phosphorylation is highly correlated with differentiation phenotype. I conclude that mTORC1 is involved in pluripotency maintenance and differentiation through cross-talk with the Wnt signalling pathway. My screen has demonstrated the power of CRISPR-Cas9-mediated screen and provided further insights in biological pathways involved in regulating differentiation. It would be interesting to explore the remaining unstudied genes for better understanding of the mechanisms underlying mESC differentiation. University of Cambridge 2018-07-21 Thesis Doctoral Doctor of Philosophy (PhD) en https://www.repository.cam.ac.uk/handle/1810/277552 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b93e8d4d-41dc-44ad-931d-16484112ac4a/download 767221d3da896e72283094641539ca73 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/06d1c8b0-e4f1-4a7f-aae7-8259cbe563bf/download 78807b91c65e349b6d15d00540843fc7 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b27a6a1a-858a-45d6-883c-ab7d2a794da0/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ Pluripotency CRISPR Genetic screen