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Researchers Use CRISPR to Destroy Pathogenic RNA Species Linked to Huntington's Disease, ALS

NEW YORK (GenomeWeb) – In a new paper published in Cell today, an international team of researchers reported using a CRISPR-Cas9-based technique to visualize and eliminate pathogenic RNA species produced by microsatellite repeat expansions (MREs) in DNA that cause dominantly inherited diseases such as Huntington's disease and amyotrophic lateral sclerosis (ALS).

The RNA-targeting Cas9 (RCas9) CRISPR system was developed by University of California, San Diego researchers in March 2016 to track specific RNA sequences and processes in vivo. At that time, they said they could use their system to visualize specific RNA molecules accumulating in stress granules — dense aggregations of proteins and RNA that form in the cytosol in response to cellular stress and have been linked to neurodegenerative disorders such as ALS. They also found that they could use Cas9 to target an mRNA without altering mRNA abundance or the amount of translated protein.

In their new study, those researchers along with collaborators in Florida and Singapore aimed to turn the RCas9 system into a diagnostic and therapeutic tool, and were able to target and destroy MRE RNAs both when exogenously expressed and in cells of patients suffering from myotonic dystrophytype 1 and 2 (DM1/2), Huntington's, andC9orf72-linked ALS (C9-ALS).

"Importantly, RCas9 reverses hallmark features of disease including elimination of RNA foci among all conditions studied, reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes," the authors wrote.

"This is exciting because we're… targeting the root cause of diseases for which there are no current therapies to delay progression," senior author Gene Yeo added in a statement.

Previous studies have established the ability of nuclease-null Streptococcus pyogenes Cas9 (spyCas9) fused to GFP (dCas9-GFP) to bind and track RNA in living cells. MRE RNAs frequently form highly localized RNA foci, the researchers wrote. Through various experiments, they found that appropriate single-guide RNAs (sgRNAs) allowed them to image the RNA foci formed by MREs in the various diseases they were studying, and that higher doses of dCas9-GFP resulted in elimination of repetitive RNA foci.

After establishing that RCas9 engages repeat expansion RNAs in overexpression models, the team then attempted to assess whether the system could also track and eliminate MRE RNAs in patient cells, and whether this could also occur at the level of DNA.

They found that sgRNAs targeting both DNA strands do not alter transcriptional dynamics of CTG repeats in patient cells, and that RNA foci elimination in DM1 patient cells was likely not due to DNA-level transcriptional disruption or destabilization of the DNA repeats. They observed a well-known hallmark of DM1 was reversed in the patient cells by RCas9 so that they resembled healthy cells.

Transcriptome-wide measurements of RNA splicing revealed more than 93 percent reversal of DM1-associated splicing pathology in patient myotubes, the researchers said, adding that myotubes cultured in vitro constitute an approximation of developing muscle tissue, so the result points to RCas9's therapeutic potential.

"This result correlates with reversal of DM1-associated splicing defects that occur in an sgRNA-dependent manner," the team wrote. "Polyglutamine diseases are linked by the presence of CAG repeat expansion translation products that are similarly reduced by the RCas9 system. Overall, these data indicate the potential of RCas9 to reverse the hallmark molecular defects associated with microsatellite repeat expansion diseases."

The researchers further indicated that their experiments showed a lower likelihood of off-target effects with RCas9 as compared to DNA-mediated CRISPR-based therapeutics.