Abstract
Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and autism. It results from expansion of a CGG nucleotide repeat in the 5′ untranslated region (UTR) of FMR1. Large expansions elicit repeat and promoter hyper-methylation, heterochromatin formation, FMR1 transcriptional silencing and loss of the Fragile X protein, FMRP. Efforts aimed at correcting the sequelae resultant from FMRP loss have thus far proven insufficient, perhaps because of FMRP’s pleiotropic functions. As the repeats do not disrupt the FMRP coding sequence, reactivation of endogenous FMR1 gene expression could correct the proximal event in FXS pathogenesis. Here we utilize the Clustered Regularly Interspaced Palindromic Repeats/deficient CRISPR associated protein 9 (CRISPR/dCas9) system to selectively re-activate transcription from the silenced FMR1 locus. Fusion of the transcriptional activator VP192 to dCas9 robustly enhances FMR1 transcription and increases FMRP levels when targeted directly to the CGG repeat in human cells. Using a previously uncharacterized FXS human embryonic stem cell (hESC) line which acquires transcriptional silencing with serial passaging, we achieved locus-specific transcriptional re-activation of FMR1 messenger RNA (mRNA) expression despite promoter and repeat methylation. However, these changes at the transcript level were not coupled with a significant elevation in FMRP protein expression in FXS cells. These studies demonstrate that directing a transcriptional activator to CGG repeats is sufficient to selectively reactivate FMR1 mRNA expression in Fragile X patient stem cells.
Highlights
Fragile X Syndrome (FXS) is an X-linked disorder affecting approximately 1 in 4,000 males and 1 in 8,000 females worldwide (Tassone et al, 2012; Hunter et al, 2014)
Transcriptional Activation of the FMR1 Gene by CRISPR-deficient Cas9 (dCas9) Fused to VP16 Activation Domains
At 48 h post transfection, we observed a significant increase in FMR1 transcript levels using the dCas9-VP64 construct with both the promoter pool and CGG guide RNAs (gRNAs) (Figure 1B) compared to a scrambled control gRNA or to cells transfected with only GFP
Summary
Fragile X Syndrome (FXS) is an X-linked disorder affecting approximately 1 in 4,000 males and 1 in 8,000 females worldwide (Tassone et al, 2012; Hunter et al, 2014). It is the leading inherited cause of intellectual disability and autism. Many FXS patients experience attention deficit hyperactivity disorder (ADHD), increased seizure susceptibility, anxiety and language difficulties. FXS results from expansion of a CGG trinucleotide repeat within the 5 untranslated region (UTR) of the fragile X gene, FMR1. FMR1 has between 25 and 40 CGG repeats
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