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

Abstract

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.

Genome Editing of Structural Variations: Modeling and Gene Correction

“…Aberrant splicing caused by expanded CTG repeats was reversed in neural stem cells derived from the iPSCs of a DM1 patient, in which exogenous poly A signal was inserted. In addition, CGG repeats have been deleted by introducing the Cas9 system into upstream of CGG repeats in ESCs and iPSCs of FXS patients ( Figure 3B) [94]. When CGG repeats were removed by this method, the FMR1 gene was reactivated in iPSCs from FXS patients.…”

“…The use of engineered nucleases enables the generation of disease models by conveniently introducing DNA repeats into a specific region [92], removing disease-causing expanded DNA repeats [93,94], or neutralizing the effect of expanded DNA repeats [95].…”

Human induced pluripotent stem cells for modelling neurodevelopmental disorders

“…Four groups have used FXS 107–109110 patient-derived hiPSCs to create proof of concept high-throughput drug discovery systems, though unfortunately the sensitivity and robustness of these screens is still poor and will require significant work. Promisingly though, Park et al 111 used a genomic engineering technique known as CRISPR/Cas (Box 3) to ablate CGG repeats in FXS patient-derived hiPSCs and thus restore expression of FMR1 mRNA and consequently FMRP protein. Unfortunately, they did not assess for any phenotypic reversal in this study.…”

Gene correction in patient-specific iPSCs for therapy development and disease modeling

“…In parallel to the HD-iPSC studies, Park and colleagues also used CRSIPR but with a different approach to delete the disease-associated CGG repeats in the 5′-UTR of the fragile X mental retardation 1(FMR1) gene in iPSCs derived from fragile X syndrome patients. Instead of using an exogenous homology donor template, the investigators took advantage of the high efficiency of Cas9 in generating DSBs and triggering NHEJ to delete the abnormal CGG repeat expansion (Park et al 2015a). In both the HD and fragile X syndrome gene correction studies, the deletion of trinucleotide repeat expansions in patient-iPSCs resulted in restoration of gene expression and reversal of susceptibility to cell death in neural stem cells derived from corrected iPSCs, suggesting that the genome-editing approach could provide alternative therapies to these neurodegenerative diseases.…”