Rescue of Fragile X syndrome by DNA methylation editing of the FMR1 [ChIP-seq]

Fragile X syndrome (FXS), the most common genetic form of intellectual disability in male, is caused by the silence of FMR1. Hypermethylation of the CGG expansion mutation in the 5’UTR region of FMR1 in FXS patients was thought to epigenetically silence FMR1. Here, we applied our previously developed DNA methylation editing tool to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/gRNA switched the heterochromatin status of the FMR1 promoter to an active chromatin status and subsequently restored FMR1 expression in FXS iPSCs. Neurons derived from methylation edited FXS iPSCs showed a similar electrophysiological property as wild-type neurons, and maintained FMR1 expression for months after engrafting into the mouse brain. Reactivation of FMR1 can be achieved in FXS neurons with demethylation of the CGG expansion. Lastly, we showed that targeted demethylation of the FMR1 promoter can reactivate FMR1 as well suggesting potential therapeutic approaches for FXS. This series includes two sets of Cas9 ChIP in FX52 iPSCs. The first set includes 3 samples: the input and IP sample from an iPSC line stably expressing dCas9-Tet1 and a guide RNA targeting FMR1 5' UTR repeat, and the IP sample from a mock iPSC line. The second set of samples include Cas9 IP from two additional lines with the same transgene expressed at a lower level, and an input sample from line 3.