CRISPR/Cas9-mediated deletion of a GA-repeat in human GPM6B leads to disruption of neural cell differentiation from NT2 cells

Abstract

Abstract The human neuron-specific gene, GPM6B (Glycoprotein membrane 6B), is considered a key gene in neural cell functionality. This gene contains an exceptionally long and strictly monomorphic short tandem repeat (STR) of 9-repeats, (GA)9. STRs in regulatory regions, may impact on the expression of nearby genes. We used CRISPR-based tool to delete this GA-repeat in NT2 cells, and analyzed the consequence of this deletion on GPM6B expression. Subsequently, the edited cells were induced to differentiate into neural cells, using retinoic acid (RA) treatment. Deletion of the GA-repeat significantly decreased the expression of GPM6B at the RNA (p < 0.05) and protein (40%) levels. Compared to the control cells, the edited cells showed dramatic decrease of the astrocyte and neural cell markers, including GFAP (0.77-fold), TUBB3 (0.57-fold), and MAP2 (0.2-fold). Subsequent sorting of the edited cells showed an increased number of NES (p < 0.01), but a decreased number of GFAP (p < 0.001), TUBB3 (p < 0.05), and MAP2 (p < 0.01), compared to the control cells. In conclusion, CRISPR/Cas9-mediated deletion of a GA-repeat in human GPM6B, led to decreased expression of this gene, which in turn, disrupted differentiation of NT2 cells into neural cells.