Reversion of FMR1 Methylation and Silencing by Editing the Triplet Repeats in Fragile X iPSC-Derived Neurons

• Fragile X iPSCs were edited with CRISPR/Cas9 to remove the CGG repeats in the FMR1 gene
• Edited FXS iPSCs showed complete ablation of the repeats and expression of FMR1 mRNA
• The FMR1 promoter in edited FXS iPSCs showed a near complete demethylation
• Edited FXS iPSCs that differentiated to mature neurons showed reactivation of FMRP

SummaryFragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from a CGG repeat expansion in the fragile X mental retardation 1 (FMR1) gene. Here, we report a strategy for CGG repeat correction using CRISPR/Cas9 for targeted deletion in both embryonic stem cells and induced pluripotent stem cells derived from FXS patients. Following gene correction in FXS induced pluripotent stem cells, FMR1 expression was restored and sustained in neural precursor cells and mature neurons. Strikingly, after removal of the CGG repeats, the upstream CpG island of the FMR1 promoter showed extensive demethylation, an open chromatin state, and transcription initiation. These results suggest a silencing maintenance mechanism for the FMR1 promoter that is dependent on the existence of the CGG repeat expansion. Our strategy for deletion of trinucleotide repeats provides further insights into the molecular mechanisms of FXS and future therapies of trinucleotide repeat disorders.

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