Screen For Genes Promoting Exit From Naive Pluripotency Based On Genome-scale CRISPR-Cas9 Knockout

The machinery regulating exit from naive pluripotency of pluripotent stem cells and the acquisition of functional cells(or tissue)are the two core issues in the field of stem cells and regenerative medicine at the present stage,which forms the first barrier determining whether stem cell and regenerative medicine research can be transformed into clinical practice.the mechanisms of the maintenance of stem cell pluripotency is well described.In contrast,it is currently unclear that how pluripotent stem cells exit from naive pluripotency(homeostasis of continuous self-renewal and proliferation)and enable eventual lineage commitment.Although endogenous repressors Tcf711,Tfe3 and the NuRD complex play an important role,but other key genes promoting exit from naive pluripotency have yet to be identified.Therefore,we perform a genome-scale CRISPR-Cas9 knockout screening in mouse embryonic stem cells(mESCs),in order to identify genes promoting exit from naive pluripotency in an unbiased manner for the first time(at the level of genome).In this project,we successfully established the model of genome-scale CRISPR-Cas9 knockout screen in mESCs for genes promoting exit from naive pluripotency.Experimental techniques used including lentiviral transfection/infection,lenti-Cas9 knockout and shRNA gene knockdown and so on,combining with data acquisition and processing methods such as pcr-resequencing,Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout and GO analysis,in the end,it is initially uncovered that Epcl and Fbxw7 may play a role in promoting exit from naive pluripotency.the present study applied the CRISPR/Cas9 genome-scale knockout technique into exploring pluripotency exiting of pluripotent stem cell for the first time and successfully established the model of genome-scale knockout screen for genes promoting exit from naive pluripotency.We used a classical differentiation model,that is,in the defined medium of mouse embryonic stem cells(N2B27),differentiation was enabled by withdrawal of the two inhibitors and LIF.While the positive genes(including TCF711 and P53)were screened out as expected,the new ones,Epcl(Enhancer Of Polycomb Homolog 1)and Fbxw7,which promoted exit from naive pluripotency,was also uncovered in the end and this provided a clue for further studying and enriching the knowledge of cell fate regulation.This paper was divided into three parts,including the establishment of screening model,the analysis and the experimental verification of screening data,which uncovered the contents and achievement of this research in turn.