An unbiased genomewide screen uncovers 7 genes that drive hematopoietic stem cell fate from mouse embryonic stem cells Available to Purchase

• CRISPR activation screening during mesodermal differentiation from mouse embryonic stem cells identifies 7 genes with HSC induction potential.

• A cocktail of 7 genes activated at the mesodermal stage induces mouse embryonic stem cell–derived multilineage repopulation and self-renewal.

Hematopoietic stem cells (HSCs) possess the ability to long term reconstitute all the blood lineages and generate all blood cell types. As such, the in vitro generation of HSCs remains a central goal in regenerative medicine. Despite many efforts and recent advancements in the field, there is still no robust, reproducible, and efficient protocol for generating bona fide HSCs in vitro. This suggests that certain regulatory elements have yet to be uncovered. Here, we present a novel and unbiased approach to identifying endogenous components to specify HSCs from pluripotent stem cells. We performed a genomewide CRISPR activator screening during mesodermal differentiation from mouse embryonic stem cells. After in vitro differentiation, mesodermal KDR⁺ precursors were transplanted into primary and secondary immunodeficient NSG mice. This approach led to the identification of 7 genes (Spata2, Aass, Dctd, Eif4enif1, Guca1a, Eya2, and Net1) that, when activated during mesoderm specification, induce the generation of hematopoietic stem and progenitor cells. These cells are capable of serial engraftment and multilineage output (erythroid, myeloid, and T and B lymphoid) in vivo. Single-cell RNA sequencing further revealed that activating these 7 genes biases the embryoid bodies toward intraembryonic development, instead of extraembryonic, increasing the number of mesodermal progenitors that can generate HSCs. Our findings underscore the importance of differentiation during the first germ layer specification to generate definitive blood stem cells.