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

• Published in: Cell reports (Cambridge) Vol. 13; no. 2; pp. 234 - 241
• Main Authors: Park, Chul-Yong, Halevy, Tomer, Lee, Dongjin R., Sung, Jin Jea, Lee, Jae Souk, Yanuka, Ofra, Benvenisty, Nissim, Kim, Dong-Wook
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13-10-2015; Elsevier
• Subjects: Cells, Cultured; CpG Islands; CRISPR-Cas Systems; DNA Methylation; Fragile X Mental Retardation Protein - genetics; Fragile X Mental Retardation Protein - metabolism; Fragile X Syndrome - genetics; Gene Silencing; Humans; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - metabolism; Neurons - cytology; Neurons - metabolism; Promoter Regions, Genetic; Trinucleotide Repeats
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2015.08.084

Fragile 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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•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
Park et al. demonstrate deletion of the extended CGG repeats in fragile X-derived induced pluripotent stem cells. The ablation of the triple repeats caused demethylation of the FMR1 promoter as well as reactivation of the gene both at the mRNA and protein levels that persisted through differentiation into mature neurons.