CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment

CRISPR/Cas9 genomic editing was used to delete the first intronic region containing six highly conserved GLl1 binding sites. The decreased GLl1 gene expression impacted GLl1 regulatory capacity within the Sonic Hedgehog signaling pathway. The result was a reduced transcriptional activation of upstream and downstream GLl1 targets, which ultimately impacted endodermal, mesodermal, and ectodermal differentiation via downregulation of key genes. AbstractGLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1 ‐edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequenc ing, quantitative‐real time PCR (q‐rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spon...
Source: Stem Cells - Category: Stem Cells Authors: Tags: Embryonic Stem Cells/Induced Pluripotent Stem Cells Source Type: research