Targeted CRISPR-Cas9 screening identifies core transcription factors controlling murine haemato-endothelial fate commitment

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 FLK1+ single cells from different genotypes ( Ctrl, Etv2 KO, Ldb1 KO, Six4 KO, Smad1 KO, Zbtb7b KO) were FACS sorted and fixed using the 10x Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (PN-1000414) and stored at -80°C. The quantity of fixed cell suspension was assessed using an automated cell counter (LUNA-FX7, Logos). Library preparation was conducted using the 10x Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples. Briefly, four fixed samples underwent probe hybridization, pooling, and loading onto the chip. Approximately 66,000 cells from the sample pools were loaded into the 10x Chromium X. Library preparation followed the manufacturer's guidelines (Guide CG000527, Rev E).