3076 – FINE-TUNING GENE EXPRESSION BY CRISPRA IN MOUSE EMBRYONIC STEM CELLS FOR SPECIFIC TISSUE INDUCTION

Abstract

Hematopoietic stem cells (HSCs) constitute the base of the hematopoietic system, giving rise to every blood cell type. Because of their clinical potential, numerous approaches have been tackled trying to generate HSCs in vitro for regenerative medicine purposes. However, due to the complexity HSCs development has, still there is not a robust way to generate bona-fide HSCs, bringing out the necessity to explore alternatives to identify genetic programmes that could lead in a robust generation of this kind of cells. In this way, gene activation by alternative CRISPR (clustered regularly interspaced short palindromic repeats) tools is a novel approach to activate endogenous genes by the simple use of gRNAs targeting promoter regions. This platform can be used in CRISPRa-based screening using library of gRNAs targeting gene promoters in a genome-wide and unbiased fashion, or alternatively can be used to activate the transcription of specific groups of genes. To test the best CRISPRa system to be used in mESCs, we generated mESCs containing the two most widely used CRISPRa systems (SunTag and SAM) that can induce a gene activation constitutively. In mESCs, SunTag induces a more robust gene expression than SAM in all the genes tested. In parallel, we have generated two inducible CRISPRa (iCRISPRa) mESC lines, VPR and iSAM. We will present the comparison of all the different CRISPRa systems in mESC and in hemogenic differentiated cells to determine the most robust iCRISPRa system in mouse embryonic stem cells differentiation system.