Rescue of Fragile X syndrome neurons by DNA methylation editing of the FMR1 gene [RNA-seq]

Fragile X syndrome (FXS), the most common genetic form of intellectual disability in male, is caused by silencing of the FMR1 gene by hypermethylation of the CGG expansion mutation in the 5'UTR region of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/sgRNA switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS. Overall design: RNA-seq of FXS iPSC and neurons derived from FXS iPSC infected with lentiviruses expressing dCas9-Tet1-P2A-tBFP (dC-T) and a mCherry-expressing sgRNA targeting CGG repeats.

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Liu XS, Wu H, Krzisch M, Wu X, Graef J, Muffat J, Hnisz D, Li CH, Yuan B, Xu C, Li Y, Vershkov D, Cacace A, Young RA, Jaenisch R