Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5′ UTR 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/single guide RNA (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 that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.

To dissect the functional significance of DNA methylation events in the human genome (), we and others have designed DNA methylation editing tools by fusion of a catalytically inactivate Cas9 with the DNA methylation modification enzymes Dnmt or Tet (dCas9-Dnmt/Tet), allowing targeted modification of DNA methylation in the mammalian genome in vitro and in vivo (). In the present study, we applied this tool to reverse the hypermethylation of CGG repeats at the FMR1 locus in multiple FXS patient-derived iPSCs. We designed a single single guide RNA (sgRNA) to guide the dCas9-Tet1 to efficiently demethylate the CGG repeats in the pathological FMR1 locus. Complete demethylation of the CGG expansion induced hypomethylation of the CpG island, increased histone H3 lysine 27 (H3K27) acetylation and H3K4 trimethylation, decreased H3K9 trimethylation at the FMR1 promoter, and unlocked the epigenetic silencing of the FMR1 gene, restoring FMRP expression in FXS iPSCs and neurons with no significant off-targeting effect. Expression of FMR1 and demethylation of its promoter in edited FXS cells were maintained for at least 2 weeks after inhibition of dCas9-Tet1 by a bacteriophage protein, anti-CRISPR type II-A 4(AcrIIA4). Epigenetic editing rescued the electrophysiological abnormalities of FXS neurons, and the reactivation of FMR1 was maintained in edited neurons in vivo following transplantation into the mouse brain. We also demonstrated that demethylation of the CGG repeats in post-mitotic FXS neurons reactivated FMR1 and reversed the spontaneous hyperactivity associated with FXS neurons. Our study provides a proof of concept that reversion of gene inactivation by epigenome editing may be a valid therapeutic strategy for disorders that involve epigenetic silencing.

The CRISPR/Cas9 system, developed for gene editing in the mammalian genome (), has been used for correcting disease-causing mutations (). Similar gene editing approaches have been used to shorten the CGG trinucleotide repeats at the 5′ UTR to partially restore FMR1 expression and normalize the physiological function of FXS patient-derived cells in the dish (), providing a proof of principle for targeted therapies that involve FMR1 reactivation. Also, 5-aza-2′-deoxycytidine treatment, inhibition of polycomb repressive complexes, and other molecular interventions for transcriptional regulation have been shown to partially reactivate FMR1 in patient fibroblast cells (), suggesting that epigenetic regulation plays a major role for FMR1 silencing in FXS.

Currently, there is no cure or treatment for FXS, likely because of the lack of a mechanistic understanding of FXS pathophysiology at the molecular and cellular level and the enormous complexity of the FXS neuronal circuitry phenotype (). Insertion of CGG repeats into the mouse Fmr1 locus did not result in DNA hypermethylation or repression of Fmr1 expression (). Although Fmr1 knockout (KO) mice partially recapitulate the neuronal hyper-excitability and excessive spinogenesis in the brain (), the FXS mouse model neither harbors the accurate genetic context nor fully recapitulates the phenotypes of FXS patients. For example, the PAK1 inhibitor and negative allosteric modulator for mGluR5 have been found to ameliorate synaptic functions in the Fmr1 KO mouse model but have variable efficacy in FXS patients (). Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived from FXS patients have been reported to properly model the hypermethylation of CGG repeats and the silencing of FMR1 (), representing a useful and complementary tool to study FXS.

Fragile X syndrome (FXS) is the most common genetic form of intellectual disability, with an incidence of one in 3,600 males. Patients with FXS display a broad spectrum of autistic phenotypes such as intellectual, cognitive, and social deficits (). These deficits are attributed to the loss of the fragile X mental retardation protein (FMRP) encoded by the FMR1 gene during brain development. FMRP is an RNA binding protein in neurons and has been shown to be a molecular brake for local protein synthesis at developing synapses and, hence, is essential for the maintenance of normal synaptic plasticity (). Indeed, loss of FMRP expression in patient-derived neurons leads to deregulated production and membrane insertion of neurotransmitter receptors and ion channels, causing synaptic hyper-excitability that affects the proper function of various neural circuits in the CNS (). A CGG trinucleotide repeat (> 200) expansion mutation at the 5′ UTR of FMR1, accompanied by DNA hypermethylation, was thought to result in heterochromatin formation at the FMR1 promoter and subsequent silencing of FMR1 expression in FXS (), but the molecular mechanism are not fully understood.

Because neurons are the most disease-relevant cell type in FXS patients, we tested whether FMR1 could be reactivated with dCas9-Tet1/CGG sgRNA in post-mitotic neurons derived from FXS iPSCs. After infection with lentiviruses expressing dC-T or dC-dT with CGG sgRNA, the mRNA level of FMR1 in dC-T/CGG sgRNA-expressing FXS neurons was restored to 45% of the one in wild-type neurons, but dC-dT/CGG sgRNA did not reactivate FMR1 ( Figure 7 B). FMRP proteins were only detected in neurons expressing dC-T/CGG sgRNA but not dC-dT/CGG sgRNA ( Figure 7 A). These results suggest that reactivation of FMR1 in post-mitotic FXS neurons is achievable by dCas9-Tet1/CGG sgRNA but less efficiently compared with FXS iPSCs ( Figures 1 B and 1C). Analysis of the CGG methylation status showed a 30% reduction in the edited neurons compared with FXS mock neurons ( Figure 7 C). BS-seq of the FMR1 promoter in these neurons showed a 20% decrease of the methylation level in the edited FXS neurons compared with FXS mock neurons ( Figure 7 D). The demethylation of the CGG repeats and subsequent demethylation of the FMR1 promoter and the reactivation of the FMR1 gene were less robust compared with that seen in edited iPSCs ( Figure 1 ). This is likely due to our inability to isolate double vector-infected neurons by FACS and the different DNA demethylation mechanism in post-mitotic neurons compared with dividing iPSCs (). Nevertheless, MEA assays revealed a rescue of the electrophysiological abnormalities in the edited FXS neurons ( Figure 7 E), suggesting that the spontaneous hyperactivity associated with FXS neurons was reversed after reactivation of FMR1 in these neurons.

(A) FX52 neurons were infected with lentiviruses expressing dC-T and the CGG sgRNA or dCas9-dTet1 (dC-dT) with the same sgRNA. Infected neurons were subject to immunofluorescence staining. Scale bar, 50 μm. Neurons were stained green for FMRP, red for Cas9, blue for DNA, and gray for MAP2.

To evaluate the rescue effects observed in methylation-edited FXS cells compared with cells lacking the CGG expansion mutation, we characterized a pair of FXS iPSC lines, including an isogenic line with CGG deletion, by CRISPR/Cas9 technology (). Both FMR1 mRNA and FMRP were restored in the CGG deletion line ( Figures 6 A–6C). The methylation level of the FMR1 promoter was reduced to 39% in the CGG deletion line ( Figures 6 D and 6E). Importantly, the spontaneous hyperactivity associated with FXS neurons was significantly reduced in CGG deletion FXS neurons, as shown in Figures 6 F and 6G. Thus, the decrease of FMR1 promoter methylation and the reversal of hyperactivity in CGG deletion FXS cells are consistent with our observations in methylation-edited FXS cells, suggesting a functional rescue of FXS-related cellular phenotypes by demethylation of the CGG expansion.

To test whether the reactivation of FMR1 in methylation-edited FXS cells is sustainable in vivo, FX52 mock- or methylation-edited neuronal precursor cells (NPCs) were labeled with GFP or red fluorescent protein (RFP) lentiviruses, respectively, and then the mixture of these two types of NPCs was injected into the P1 mouse brain for subsequent analysis 1 or 3 months after transplantation ( Figure 5 G). Immunofluorescence staining of the mouse brain sections showed that 56% and 57% of the edited FX52 neurons (RFP-positive) in 1- and 3-month post-transplanted mice, respectively, expressed FMRP, whereas FX52 mock neurons (GFP-positive) were negative for FMRP expression ( Figure 5 H), suggesting that FMR1 reactivation can be maintained in vivo after transplantation.

To evaluate the effect of FMR1 reactivation on the rescue of FXS-related cellular phenotypes, post-mitotic neurons were derived from the methylation-edited FX52 iPSCs, as shown in Figure 5 A, with a well-established differentiation protocol (). Gene expression analysis of lineage-specific markers suggested comparable differentiation states between wild-type and mutant neural cultures ( Figure 5 B). The expression level of FMR1 in neurons expressing dC-T/CGG sgRNA was 82% of the one in wild-type neurons, and FMR1 remained silent in the neurons expressing dC-dT/CGG sgRNA ( Figure 5 C), suggesting that differentiation of edited FXS iPSCs did not affect FMR1 reactivation. Immunohistochemistry confirmed that FMRP remained expressed in the edited neurons ( Figure 5 D). Genome-wide methylation analysis by ChIP-BS-seq using neurons derived from FX52 iPSCs expressing dC-T/CGG sgRNA or dC-dT/CGG sgRNA allowed comparison of methylation levels at 670 binding sites and identified 43 sites with a change of methylation larger than 10%, including a 38% decrease of methylation at the FMR1 locus ( Table S2 ). Nevertheless, transcriptome analysis of FX52 mock neurons and edited neurons outlined FMR1 as the most upregulated gene (481-fold) and showed either no change or a minor change (4-fold) of expression for the 41 genes and a 9-fold change for the RGPD1 gene associated with a larger than 10% methylation change, highlighted with red dots in Figure 5 E, suggesting a specific reactivation of FMR1. To examine the electrophysiological properties of methylation-edited FXS neurons, a multi-electrode array (MEA) assay was performed with wild-type WIBR1 neurons and FX52 mock-, dC-T/CGG sgRNA-, and dC-dT/CGG sgRNA-expressing neurons. As shown in Figure 5 F, the significantly higher firing rate of FX52 neurons was reduced to the levels of wild-type neurons in dC-T/CGG sgRNA-expressing neurons but not in neurons expressing dC-dT/CGG sgRNA. These results suggest that reactivation of FMR1 reversed the spontaneous hyperactive phenotype of FXS neurons.

(H) Representative confocal micrographs of cells engrafted in the mouse brain 1 month post-transplantation. Scale bar, 50 μm. Cells were stained red for RFP, green for GFP, magenta for FMRP, and blue for DNA. A total of 496 RFP-positive neurons and 149 GFP-positive neurons from three mice after 1 month were counted. 56% ± 9% of RFP neurons were FMRP-positive, whereas all GFP-positive neurons were FMRP-negative. A total of 203 RFP-positive neurons and 117 GFP-positive neurons from two mice after 3 months were counted. 57% ± 3% of RFP neurons were FMRP-positive, whereas all GFP positive neurons were FMRP-negative.

To gain insight into the kinetics of FMR1 reactivation by methylation editing, we performed a time course experiment to monitor the expression of FMR1. As shown in Figure 4 A, the expression of FMR1 was first detected at the 9-day time point after infection with dCas9-Tet1/CGG sgRNA lentiviruses and peaked around 3 weeks, accompanied by demethylation of the FMR1 promoter ( Figure 4 D). To examine the persistence of methylation editing, we used AcrIIA4, a previously described inhibitor of Cas9/dCas9 (), to inactivate dCas9-Tet1. Expression of AcrIIA4 blocked the binding of dCas9-Tet1 to the FMR1 locus, as shown in Figure 4 B. We observed that FMR1 expression and demethylation of its promoter were maintained in the presence of AcrIIA4 for at least 2 weeks ( Figures 4 C and 4D), suggesting that constitutive presence of dCas9-Tet1 at the CGG repeats may not be required to sustain FMR1 reactivation through cell division.

(C) Cells on day 23 (labeled as Meth-edited) in (A) were infected with a lentivirus expressing AcrIIA4 and harvested for qPCR analysis at various time points. Shown are the mean FMR1 (red bar) and AcrIIA4 (blue bar) expression levels relative to the levels in the CGG deletion FX52 iPSC line (CGGΔ) and the cells on day 3 after AcrIIA4 infection, respectively (± SD of two biological replicates).

(B) 293T cells were transfected with dC-T alone, with dC-T/CGG sgRNA, or with dC-T/CGG sgRNA and AcrIIA4. The cells were subject to an anti-Cas9 ChIP experiment, and the enrichment of dC-T at the FMR1 and BDNF loci was examined by qPCR analysis. Shown is the mean of relative binding normalized to the input ± SD from three biological replicates.

(A) FX52 iPSCs were infected with lentiviruses expressing dC-T and a CGG sgRNA and harvested for qPCR analysis of FMR1 expression at various time points. Shown is the mean expression level relative to that in a CGG deletion FX52 iPSC line (± SD of two biological replicates). This CGG deletion FX52 iPSC line was previously reported to restore FMR1 expression ().

As shown previously, the FMR1 promoter region in FXS patient cells is in a heterochromatic conformation with DNA hypermethylation, decreased histone acetylation and H3K4 trimethylation, and increased histone H3K9 trimethylation (). To dissect the molecular mechanism of FMR1 reactivation by dCas9-Tet1/CGG sgRNA-mediated demethylation of the CGG repeats, we examined the epigenetic state of the FMR1 promoter by ChIP-seq assays with antibodies against RNA polymerase II (Pol II), histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 27 acetylation (H3K27Ac), H3K27me3, and H3K9me3. As shown in Figure 3 A, Pol II was recruited to the FMR1 promoter in the methylation-edited cells but not in the cells expressing dCas9-dTet1/CGG sgRNA. The active chromatin marks H3K4me3 and H3K27Ac were localized to the promoter regions in the edited cells, with the repressive marker H3K9me3 being reduced, suggesting a switch of the heterochromatin state of the FMR1 promoter to an active chromatin conformation after demethylation of the CGG repeats. Two control loci, including POU5F1 and MYOD1, showed no detectable change for these marks in edited cells. Genome-wide analysis of Pol II occupancy showed that FMR1 is the most upregulated gene in the edited cells, whereas the 28 genes with a change of methylation of larger than 10% showed either no change or a minor change of Pol II occupancy (less than 3-fold), except GSE1 with a 5-fold change ( Figure 3 B). Nevertheless, the expression level of GSE1 did not change in the edited cells ( Table S3 ). These data confirm a selective activation of FMR1 by dCas9-Tet1/CGG sgRNA. Genome-wide analysis of histone H3K4me3 distribution again highlighted the effect at the FMR1 locus with either no change or a minor change (less than 3-fold) for all 28 genes ( Figure 3 C). Our observations support the role of DNA methylation of the CGG repeats as the major epigenetic block in silencing of FMR1 and argue that demethylation of these repeats is sufficient to rebuild an active chromatin status for the FMR1 promoter, allowing its reactivation.

(B and C) Genome-wide Pol II occupancy (B) and histone H3K4me3 occupancy (C) of mock and edited FX52 iPSCs. Red dots highlight the 28 genes associated with a change of methylation larger than 10%, as described in Figure 2 E. FMR1 is labeled with a green dot. Promoters with a 3-fold or more change in factor occupancy are plotted as blue circles.

The presence of the CGG sgRNA targeting sequence GGCGGCGGCGGCGGCGGCGGNGG in other genomic loci raises concerns regarding off-target effects of the dCas9-Tet1/sgRNA system used. We examined the genome-wide binding sites for dCas9-Tet1 with CGG sgRNA by an anti-Cas9 chromatin immunoprecipitation sequencing (ChIP-seq) experiment using three FX52 iPSC lines expressing different levels of dCas9-Tet1 generated by lentiviral transduction with different viral titers. We observed that the restoration level of FMR1 decreased from 90% to 20% when the expression level of dCas9-Tet1 was reduced from 100% to 21% ( Figure 2 A), whereas reduction of CGG sgRNA did not affect the level of FMR1 reactivation ( Figure S1 C), suggesting that dCas9-Tet1 rather than sgRNA was the limiting factor. The number of genome-wide dCas9-Tet1 binding sites was also decreased when the expression level of dCas9-Tet1 was reduced ( Figure 2 B). To examine possible off-target effects in more detail, we chose FX52 iPSC line 1 for further analysis because it has the highest number of dCas9-Tet1 binding sites among the three lines in which 90% of the FMR1 mRNA level was restored ( Figure 2 A). Among these binding sites, we first analyzed the top 6 candidate loci overlapping with methylated promoter regions according to a hESC/iPSC methylome study reported previously () and showing the highest binding affinity of dCas9-Tet1 in ChIP-seq ( Figure 2 B). BS-seq showed a 20% and 30% reduction of methylation levels for SHCBP1L with one CGG sgRNA targeting site and RGPD1 with 6 targeting sites, respectively, but no detectable methylation changes for the other four genes ( Figure 2 C). The level of demethylation in SHCBP1L and RGPD1 loci likely correlates with the number of dCas9-Tet1/CGG sgRNA targeting sites. Gene expression analysis by qPCR showed either undetectable or minor changes with a 2.2-fold upregulation of SHCBP1L as the highest level of change ( Figure 2 D). To evaluate the methylation editing at a genome-wide level, we performed an anti-Cas9 ChIP-BS-seq experiment using FX52 iPSCs expressing dCas9-Tet1/CGG sgRNA (line 1 in Figure 2 A) or dCas9-dTet1/CGG sgRNA. The result of this experiment allowed comparison of methylation levels at 1,018 dCas9-Tet1 binding sites between these two lines ( Figure 2 E). 29 loci showed a change of methylation larger than 10%, with the FMR1 locus displaying the most significant methylation decrease (85%) in edited cells. Importantly, RNA sequencing (RNA-seq) analysis of mock- and methylation-edited FX52 cells showed no significant change in overall gene expression (a correlation coefficient of 0.99; Figure 2 F). In contrast to the 1,500-fold upregulation of FMR1 expression, either no change or a minor change (maximal 4-fold) of expression was detected for the 28 genes with a change of methylation larger than 10% identified by ChIP-BS-seq. These data suggest that the off-target effect of dCas9-Tet1/CGG sgRNA on the DNA methylation and transcription levels is minimal and could be further minimized by titration of the expression level of dCas9-Tet1.

To test whether demethylation of the hypermethylated CGG repeats can reactivate FMR1, we infected FX52 iPSCs, a previously described FXS iPSC line () containing about 450 CGG repeats in the 5′ UTR of FMR1, with lentiviruses expressing dCas9-Tet1(dC-T)-P2A-tag blue fluorescent protein (tBFP) and an mCherry-expressing sgRNA targeting the “GGCGGCGGCGGCGGCGGCGGNGG” sequence (CGG sgRNA), and collected cells expressing both vectors by fluorescence-activated cell sorting (FACS) ( Figure 1 A). As shown in Figure 1 B, the expression level of FMR1 mRNA in cells with dC-T/CGG sgRNA was restored to 90% of the one in wild-type WIBR1 human embryonic stem cells (hESCs), whereas cells expressing a catalytically dead Tet1 (dC-dT) and the same sgRNA did not reactivate FMR1. Further, FMRP expression was restored in dC-T/CGG sgRNA-expressing FX52 iPSCs to 73% of the wild-type level in WIBR1 cells, as shown by immunofluorescence staining and western blotting ( Figures 1 C and 1D). Methylation analysis of the CGG repeats showed a significant reduction of methylation from 100% in mock FX52 iPSCs to 4% in dC-T/CGG sgRNA-expressing FX52 iPSCs ( Figure 1 E), and bisulfite sequencing (BS-seq) of the FMR1 promoter showed a robust demethylation after infection with lentiviral dC-T/CGG sgRNA vectors ( Figures 1 F and 1G). These data indicate that dCas9-Tet1 targeted by the CGG sgRNA can efficiently demethylate the CGG repeats in FX52 iPSCs, resulting in subsequent demethylation of the FMR1 promoter and reactivation of FMR1 both on the transcriptional and translational levels. Two additional FXS iPSC lines with more than 450 CGG trinucleotide repeats () were also used to test the reactivation of FMR1 by dCas9-Tet1/CGG sgRNA. As shown in Figures S1 A and S1B, FMR1 was reactivated after lentiviral transduction of dCas9-Tet1 and CGG sgRNA in both cell lines. FMR1 was reactivated to ∼60% and ∼30% of the level seen in WIBR1 wild-type cells, respectively. The different levels of FMR1 expression are likely due to variable vector expressions in these particular experiments (see analysis in Figure 2 A). We conclude that targeted demethylation of the CGG repeat expansion by dCas9-Tet1/CGG sgRNA can reactivate FMR1 in multiple FXS patient-derived iPSCs. The FX52 iPSC line was used for further studies.

(F) RNA-seq of the mock and dCas9-Tet1/CGG sgRNA-expressing FX52 iPSC line 1 described in (A). Red dots highlight the 28 genes with a change of methylation larger than 10% identified in (E). FMR1 is labeled with a green dot. The dashed red lines mark the 4-fold difference between the samples.

(E) Anti-Cas9 ChIP-BS-seq of FX52 iPSCs expressing dCas9-Tet1/CGG sgRNA (line 1 in A) or dCas9-dTet1/CGG sgRNA. 28 binding sites with a change of methylation larger than 10% are labeled in red; FMR1 is labeled in green. The diameter of a circle is in proportion to the number of CGG sgRNA target sites; binding sites with mismatched target site are indicated by the smallest circles. Blue lines of circles indicate binding sites overlapping with a promoter region. The dashed lines mark the 10% methylation difference between samples.

(C) BS-seq and pyrosequencing (Pyro-seq) of the 6 top off-target candidate gene loci that overlapped with methylated promoter regions according to a hESC/iPSC methylome study reported previously () and showed the highest binding affinity of dCas9-Tet1 in (B). The CGG sgRNA target site (GGCGGCGGCGGCGGCGGCGGNGG) is illustrated with a red hexagon. The mismatch target site is illustrated with a yellow dot. CpGs are indicated by vertical bars. Shown is the mean percentage ± SD of two biological replicates.

(C) FX52 iPSCs were infected with different ratios of lentiviruses expressing dCas9-Tet1-P2A-BFP (dC-T) and a CGG sgRNA. Infected cells were harvested for qPCR analysis of FMR1 expression at various time points. Shown is the mean of relative percentages to the one in a CGG deletion FX52 iPSC line ± SD of two biological replicates.

(B) Another previously reported FXS iPSC line (SW) with more than 450 CGG repeats was used to examine FMR1 expression as did in B. The expression level of FMR1 were quantified by qPCR analysis and compared to an isogenic line with shortened CGG repeat expansion, and shown as the mean of relative percentages to the one in WIBR1 hESCs ± SD of two biological replicates.

(A) One previously reported FXS iPSC line (135.3) with more than 450 CGG repeats was infected with lentiviruses expressing dCas9-Tet1-P2A-BFP (dC-T) with a mCherry expressing sgRNA targeting the CGG repeats “GGCGGCGGCGGCGGCGGCGGNGG” (CCG sgRNA) or lentiviruses expressing dCas9 fused with a catalytically dead Tet1 (dC-dT) with the same sgRNA. Double positive (BFP+; mCherry+) cells were isolated by FACS after infection. The expression level of FMR1 were quantified by qPCR analysis and compared to an isogenic line with shortened CGG repeat expansion, and shown as the mean of relative percentages to the one in WIBR1 hESCs ± SD of two biological replicates.

(D) The cells in (B) were subjected to western blot analysis. The protein level of FMRP was quantified by ImageJ and is shown as the mean of relative percentages to the one in WIBR1 hESCs ± SD of two biological replicates.

(B) A previously reported FXS iPSC line (FX52) was infected with lentiviruses expressing dCas9-Tet1-P2A-BFP (dC-T) with an mCherry-expressing sgRNA targeting the CGG repeats GGCGGCGGCGGCGGCGGCGGNGG (CGG sgRNA) or lentiviruses expressing dCas9 fused with a catalytically dead Tet1 (dC-dT) with the same sgRNA. Double positive (BFP+; mCherry+) cells were isolated by FACS after infection. The expression level of FMR1 was quantified by qPCR analysis and is shown as the mean of relative percentages to the one in WIBR1 hESCs ± SD of three biological replicates.

Discussion

In this study, we applied recently developed DNA methylation editing tools to reverse hypermethylation of the CGG repeats in the FMR1 locus. An iPSC-based FXS model was chosen because it recapitulates the hypermethylation of CGG repeat expansion and epigenetic silencing of FMR1. Our results demonstrate that targeted demethylation of the CGG repeats reactivated FMR1 in multiple FXS iPSCs as well as in in vitro-derived FXS neurons. Demethylation of the CGG repeats resulted in conversion of the heterochromatic state to an active state of the non-targeted upstream FMR1 promoter. Thus, our data provide the first direct evidence that demethylation of the CGG repeats is sufficient to reactivate FMR1. Importantly, methylation editing reversed the abnormal electrophysiological phenotype of FXS neurons, and the expression of FMRP in edited neurons was maintained in vivo after transplantation into the mouse brain. To address potential off-target effects associated with the dCas9-Tet1/CGG sgRNA system, we performed a genome-wide survey to identify binding sites of dCas9-Tet1 with CGG sgRNA and examined the methylation levels of these sites. ChIP-BS-seq revealed a small set of sites with changes in methylation larger than 10%, and RNA-seq analysis showed no major off-target effects on transcriptional levels of these genes, suggesting a highly specific effect by targeted methylation editing.

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Krzyzosiak W.J. Cellular toxicity of expanded RNA repeats: focus on RNA foci. Trinucleotide repeat expansions play a pathological role in several neurological, neurodegenerative, and neuromuscular disorders such as FXS, Huntington’s disease, spinocerebellar ataxia, and myotonic dystrophy (). Repeats can elicit toxicity through a series of overlapping pathogenic cascades, including gain of function or loss of function at the protein and/or RNA level(s). Silencing of FMR1 with more than 200 CGG repeats is considered the cause for FXS. However, carrier patients with 55–200 CGG repeats have overproduction of FMR1 mRNA but reduction of FMRP protein and develop fragile X-associated tremor and ataxia syndrome (FXTAS) with some features of the FXS phenotype, including progressive cerebellar tremor and ataxia, cognitive impairment, mild parkinsonian symptoms, and brain atrophy (). The repeat-associated non-adenosine uridine guanosine (AUG) (RAN) translation of a transfected CGG repeat-containing construct has been shown to generate toxic neuronal proteins (). Also, RNA inclusions were found in a small subgroup (6%–11%) of FXTAS nuclei (). Restoration of FMRP protein and rescue of FXS cellular phenotypes after reactivation of FMR1 argue that the cellular toxicity assumed for CGG repeat-containing FMR1 RNA is likely to be minimal in methylation-edited FXS cells within a short period of culture time. It remains an open question whether the reactivated FMR1 in FXS cells results in RAN and/or forms intranuclear RNA inclusions, but our tool provides a unique experimental system to further investigate the toxicity of CGG repeats in neurons.

Accurate and efficient targeting with a controllable off-target effect is the ultimate goal for gene therapy and epigenome editing toward therapeutic intervention. To examine the potential off-target effect of dCas9-Tet1/CGG sgRNA, we performed a series of genome-wide analyses of methylation-edited FXS cells. Compared with the robust reduction in methylation of the FMR1 locus (84% decrease), a global methylation analysis of FXS cells expressing dCas9-Tet1/CGG sgRNA or dCas9-dTet1/CGG sgRNA by ChIP-BS-seq revealed that only a small set of binding loci (28 of 1,018 sites in edited FXS iPSCs and 42 of 670 sites in edited FXS neurons) changed the level of methylation by more than 10%. The high methylation editing specificity and efficiency of dCas9-Tet1/CGG sgRNA are likely due to the repetitive nature of the CGG repeats present in the FMR1 locus in FXS cells, which allows for enrichment of the dCas9-Tet1 machinery on the condensed CGG repeats for efficient methylation editing. Importantly, RNA-seq revealed either no change or minor changes of expression for the genes associated with these sites, suggesting a highly specific effect on FMR1 reactivation by methylation editing. Nevertheless, off-target binding of dCas9-Tet1 is a concern. We found that the level of dCas9-Tet1 vector expression was positively correlated with both the expression level of FMR1 and the number of off-target binding sites of dCas9-Tet1, suggesting that the dCas9-Tet1/CGG sgRNA is a tunable methylation editing tool that allows restoration of FMR1 at different expression levels, with a potentially controllable off-target effect to safely rescue FXS phenotypes. Furthermore, we observed that reactivation of FMR1 and demethylation of its promoter did not require constitutive editing by dCas9-Tet1 and was maintained for at least 2 weeks after inactivation of dCas9-Tet1. Another strategy to minimize off-target binding would be to target the dCas9-Tet1 to the FMR1 promoter instead of the CGG repeats because targeting the promoter would allow for designing unique sgRNAs and reducing potential off-target binding of dCas9-Tet1.

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Foster T.C.

Bloom D.C. Fragile X mental retardation protein replacement restores hippocampal synaptic function in a mouse model of fragile X syndrome. Whether the FXS phenotype can be reversed postnatally is unknown. It has been shown that re-expression of FMRP by AAV-based gene delivery could partially rescue the neuronal phenotype in Fmr1 mutant mice, suggesting that the functional deficits may be at least partially reversible (). We have demonstrated that restoration of FMR1 can be achieved in in vitro-derived FXS neurons by dCas9-Tet1/CGG sgRNA-mediated methylation editing. Electrophysiological analysis of the edited neurons with 45% restoration of FMR1 showed a similar behavior as wild-type neurons, indicating that the FXS cellular phenotype is likely reversible in neurons and that full restoration of FMR1 may not be necessary for a functional rescue, opening a potential therapeutic window for the treatment of FXS.

In summary, our study demonstrates that demethylation of the CGG repeats is sufficient to reactivate FMR1. Thus, methylation editing is a valid strategy to reactivate FMR1 and to rescue the FXS-related cellular phenotypes, providing a proof-of-concept paradigm to study disease-associated abnormal DNA methylation events. This method for epigenome editing can be easily applied to examine the causality of disease-associated DNA methylation events and evaluate the consequences after targeted reversal of the DNA methylation status, holding great potential for future research of novel therapies.