Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington’s disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), 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. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.
Normally, CRISPR uses a slicing protein called Cas9 that recognizes and chops up the desired DNA, eliminating a mutated gene. Yeo and his team modified Cas9 to leave DNA alone and instead bind to and cut problematic RNA.
When tested in the lab, Yeo’s CRISPR tool obliterated 95 percent or more of these RNA knots in cells harboring Huntington’s disease and a type of ALS.
The researchers also tested the approach on a form of inherited muscular dystrophy, called myotonic dystrophy. They were able to eliminate 95 percent of faulty RNAs in muscle cells taken from patients. After they applied CRISPR, the once-diseased cells resembled healthy ones. Yeo thinks more than 20 genetic diseases that are caused by toxic RNA repeats could potentially be treated this way.
Knocking down these RNAs is only temporary, though. RNA constantly regenerates, so its level in cells eventually rebounds back to normal after a few days to a week.
So Yeo is designing a virus capsule to carry the CRISPR machinery to the right cells. These viral delivery shuttles would allow the Cas protein to stick around in a person’s cells longer—ideally for years, turning Cas into a mini-arsenal to keep unruly RNA at bay.