多能性的维持与退出需要转录调控因子、表观遗传修饰因子和细胞外信号通路的相互作用。目前已经有大量研究是针对于染色质结合蛋白的，然而对于RNA 结合蛋白（RBPs）所发挥的作用了解的不多。我们在前期工作中通过生物信息学分析获得了13个可能参与ESC退出多能性的RBPs。随后我们利用CRISPR-Cas9技术在单倍体mESC中对这些RBPs进行了敲除，并利用分化实验对RBPs缺失后mESC的分化能力进行了检测，其中RBP-X缺失后分化缺陷表型最为显著，已有研究表明RBP-X可以通过调控可变剪接影响B细胞分化，但在mESC中的作用机制尚未明确。在本研究中，我们将RBP-X作为目标，为排除细胞背景影响，在二倍体mESC中对其进行敲除，利用RNA-seq及CLIP-seq等实验手段找到RBP-X调控的靶RNA以及影响的RNA加工过程，揭示其在ESC分化过程中的作用机制。

Pluripotency maintenance as well as the exit and re-acquisition of pluripotency by de-differentiation and somatic cell reprogramming, require the interplay of transcriptional regulators, epigenetic modifiers, and extracellular signaling pathways. Tremendous efforts have been directed toward studying chromatin binding proteins such as DNA binding transcription factors and chromatin modifying proteins However, much less is known about the roles of RNA-binding proteins (RBPs) in pluripotency, differentiation, and reprogramming. RBPs participate in every step of RNA biology, from transcription, splicing, and polyadenylation to RNA modification, transport, translation, and turnover. RNAs in cells are associated with RNA-binding proteins (RBPs) to form ribonucleoprotein (RNP)..Here, by functional screening some pre-selected RBPs in ES cells, we identified a few critical RBPs required for exit from self-renewal and further explored their molecular mechanisms. Firstly, 13 candidate RBPs were picked by bioinformatics analysis based on the mouse ES cell RBPs database and published literatures. And then we disrupted these candidates by using CRISPR-Cas9 system in mouse haploid ES cells and got the mutant cell lines respectively. The differentiation ability of these mutant cell lines were evaluated through embryoid body formation and withdraw LIF from the medium. RBP-X RBP-null cell clones showed significant differentiation defect compared with wide-type ES cells..RBP-X is a tissue-specific hnRNP, which was identified as a regulator of CD45RA to CD45RO switching during memory T-cell development. Since then, RBP-X has emerged as a critical regulator of lymphocyte homeostasis and terminal differentiation, controlling alternative splicing or expression of critical genes for the lymphocytes development. There was no evidence of functional roles about RBP-X in mouse ES cells. .In this study we will disrupt RBP-X in diploid mESC, We will perform RNA-seq and CLIP-seq by using the EB samples of RBP-X-null ESCs and wide-type ESCs, and compare the change of the whole transcriptome and alternative splicing events. We will focus on the influence of RBP-X on alternative splicing events. And finally reveal the functional role RBP-X in ESC Exit from Pluripotency.