Targeted CRISPR-Cas9 screening identifies core transcription factors controlling murine haemato-endothelial fate commitment [RNA-seq]

During development, blood generation begins in the yolk sac with the differentiation of haemato-endothelial mesoderm generating haematopoietic progenitors. This study aimed to identify the crucial molecular regulators of haemato-endothelial mesoderm formation and to extend our knowledge of the process in an unbiased way. We employed a murine embryonic stem cell model that recapitulates embryonic blood development, and performed targeted CRISPR-Cas9 knock out screens focusing on transcription factors and chromatin regulators, which highlighted the transcription factors Smad1, Ldb1, Six4 and Zbtb7b. Embryonic stem cells lacking these regulators gave rise to mesodermal subsets with a defined lineage differentiation bias, while transcriptome analysis of these cells uncovered the precise impact of each factor on gene expression in the developing mesoderm. Our study reveals novel molecular pathways governing mesodermal development crucial to allow endothelial and haematopoietic lineage specification and paves the way for future advances in haematopoietic stem cell applications. Overall design: Viable single cells were FACS sorted into 384-well cell capture plates (Single Cell Discoveries, Netherlands), in which each well contained 50 nl of barcoded primers and 10 µl of mineral oil (Sigma M8410). After sorting, plates containing cells were centrifuged at 1500x g for 1 minute then placed on dry ice before storage at -80° C. Single-cell RNA sequencing was performed by Single Cell Discoveries according to an adapted version of the SORT-seq protocol57