Dealing with microsatellite disorders has been as difficult as shooting down actual satellites, but thanks to dCas9 its gotten a lot easier. The CRISPR/Cas9 system has proven to be much more than just a pair of scissors. For instance, CRISPR interference (CRISPRi) uses deactivated Cas9 (dCas9) to create site-specific steric hinderance to perturb gene expression. A new study from Eric Wang’s lab at the University of Florida examines several dCas9 approaches to treat repeat expansion disorders.
The talented has previously examined various approaches to treat conditions such as myotonic dystrophy, a muscle wasting disorder. Type 1 (DM1) is caused by a (CTG)n triple repeat expansion in DMPK 3’UTR, while type 2 (DM2) is caused by a (CCTG)n repeat expansion in CNBP intron 1. These expanded transcripts sequester specific proteins from their RNA targets leading to abnormal RNA splicing stability. Researchers believe that the efficiency of transcription through the expanded repeats is decreased relative to non-repetitive sequences. If this is true, further impairing the transcription of the expanded allele could effectively silence it. The authors attempted this by targeting the expanded alleles from various disorders in a repeat-length dependent manner using dCas9. This would result in premature RNA polymerase termination and nascent transcript turnover of the expanded allele, while leaving the normal allele unaffected.
To do this, the authors used dCas9 and various guide RNAs to target microsatellite repeat sequences for DM1, DM2, and other repeat disorders. They hypothesized that the dCas9 proteins bound to the repeat would be sufficient to block transcription. The team optimized this system by testing various guide RNA/PAM sequence pairs for efficacy in silencing repeats of increasing length expressed from plasmids.
Here’s what they found:
- Using plasmids in HeLa cells, only 1 of the 4 guide RNA/PAM pairs tested reduced expression of the expanded allele, and did so in a repeat-length dependent manner. This was repeated when these repeats were incorporated into the Hela cell genome
- Targeting of (CCTG)n repeat expansion also blocked expression of the expanded gene from a plasmid in
- In DM1 patient-derived cell lines, dCas9 treatment rescued repeat expansion phenotypes including reducing toxic RNA foci, and rescued splicing deficits
- Using an ex vivo muscle culture from a DM1 mouse model, dCas9 application led to a ~50% restoration of muscle function
The team found that the longer the repeat, the better the dCas9-based silencing worked. This allows the non-expanded allele to be expressed normally, a major challenge for other approaches. Going forward, systems such as this may be useful as gene therapy for repeat disorders where pathogenesis is dependent on very long repeat lengths.
Get the full message over at Molecular Cell, November 2017