The call for a rescue operation often makes humanity turn to its latest and greatest tools and equipment. Whether it’s firefighters with water jetpacks or cutting-edge epigenetic editing technology, threats to human kind don’t have it easy these days. In their latest report, the epigenetic emergency response team from the labs of Rudolf Jaenisch and Richard Young at MIT applied their in vitro and in vivo DNA methylation editing toolbox to rescue human neurons with Fragile X Syndrome (FXS); the leading genetic cause of intellectual disabilities in males.
FXS is caused by a CGG expansion in the 5’ UTR of the FMR1 gene, where DNA hypermethylation of the expansion represses gene expression. To reverse this repressive pattern and gain insight into the epigenetic mechanisms of FXS, the talented team invoked some precision DNA demethylation via dCas9-Tet1.
Here’s what happened:
- Lentiviral transduction of patient-derived induced pluripotent stem cells (iPSCs) with dCas9-Tet1 and a sgRNA targeting the FMR1 repeat successfully demethylated the CGG repeats to induce gene (FMR1) and protein (FMRP) expression
- Anti-Cas9 ChIP-BS-seq and RNA-seq analysis demonstrated minimal off-target effects, as the largest changes occur at the desired target site
- The off-target effects can be further minimized by titrating the dCas9-Tet1 expression level
- ChIP-seq of the edited patient-derived iPSCs revealed that the non-targeted upstream FMR1 promoter switches from a silenced heterochromatic state to an active chromatin state
- This analysis revealed an enrichment of RNA polymerase II and active chromatin marks (H3K4me3 and H3K27Ac), but a reduction in the levels of repressive chromatin marks (H3K9me3)
- Time course experiments revealed that:
- Expression of FMR1 is first detected 9 days after transfection, with peak expression at 3 weeks
- DNA demethylation and gene expression persist for at least 2 weeks after using anti-CRISPR type II-A 4 (AcrIIA4) to block binding of dCas9-Tet1 to the FMR1 target site, suggesting that constitutive expression of the dCas9-Tet1 system may not be required
- Derivation of post-mitotic neurons from the edited FXS iPSCs revealed that editing rescued the hyperactive electrophysiological phenotype
- Moving in vivo, the team engrafted neuronal precursor cells (NPCs) derived from the edited FXS iPSCs into post-natal day 1 mouse brains, where they observed maintained FMR1 expression for up to 3 months
- Finally, the team demonstrated successful editing of neurons derived from FXS iPSCs
First Author Shawn Liu shares, “We showed that this disorder is reversible at the neuron level. When we removed methylation of CGG repeats in the neurons derived from fragile X syndrome iPS cells, we achieved full activation of FMR1.”
Senior Author Rudolf Jaenisch concludes, “These results are quite surprising — this work produced almost a full restoration of wild type expression levels of the FMR1 gene. Often when scientists test therapeutic interventions, they only achieve partial restoration, so these results are substantial. This work validates the approach of targeting the methylation on genes, and it will be a paradigm for scientists to follow this approach for other diseases.”
Go learn how dCas9-Tet1 can rescue your experiments over at Cell, February 2018.