Defining Essential Genes for Neuroectoderm

Human embryonic stem cells (hESCs) have the potential to differentiate to every cell of the body. Understanding the developmental processes of these cells will contribute to the design of novel cell therapies and the generation of better models for human diseases. The complete gene networks that are essential for these early differentiation events are yet to be determined. Here, we aimed to identify the subset of genes that are essential for the differentiation of hESCs into the neuroectodermal lineage. To this end, we have utilized a genome-wide loss-of-function library covering all 18,000 coding genes, generated in haploid hESCs by CRISPR/Cas9 technology and an established differentiation protocol for neural progenitor cells (NPCs). We have shown that 2.9% of all genes were significantly depleted within the NPC population, but not in the hESC population suggesting that these genes are specifically essential for the development of NPCs. In contrast, we have also demonstrated that 2.8% of the genes were enriched within the NPC population, implying that these genes inhibit NPC development. We have identified two close homologs from the homeobox-containing transcription factor family, namely POU3F2 and POU3F3, as essential genes for NPC development. In order to validate our screen results, we have utilized CRISPR/Cas9 mutagenesis and generated a POU3F2 null hESC line, which, upon differentiation to NPCs, exhibited downregulation of neural differentiation markers. Our work maps essential genes for the formation of neuroectoderm and may serve as reference for future studies on human neural development.