Transcriptomic analysis of NPCs and neurons derived from WT and FMR1-KO hPSCs. (A) Schematic diagram of generation of isogenic FMR1 KO hPSCs using a seamless CRISPR-Cas9 strategy. (B) Sanger sequencing shows a 7-bp deletion in the FMR1 gene and a premature stop codon of FMR1 KO cells. (C) Western blot confirming knockout of FMR1 in FMR1 KO hPSCs, GAPDH as a loading control. hPSC lines in red were used for experiments. (D) Volcano plot of gene expression in WT and FMR1 KO dNPCs. (E) GO enrichment of the 277 genes up-regulated in FMR1 KO dNPCs. (F) GO enrichment of the 86 genes down-regulated in FMR1 KO dNPCs. (G) Violin and box plots of up-regulated (up) and down-regulated (down) genes in KO dNPCs with respect to their expression levels in dNPCs and dNeurons (Box plot: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range). (H) Representative images of EdU incorporation assay of dNPCs derived from H1 WT and KO hPSCs (scale bar, 50 µm). (I) Quantitative analysis of the percentage of EdU+ dNPCs (mean ± SE; dots and lines show batches of differentiation; P = 0.02, paired t-test, two tails).
