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Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9
Ranjan Batra1, David A Nelles1, Elaine Pirie1
1Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA; Stem Cell Program, University of California at San Diego, La Jolla, CA, USA; Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA, USA.
Abstract:
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.
Insights
Scientists developed a CRISPR system to target and eliminate toxic RNA in repeat expansion diseases. This RNA-targeting Cas9 (RCas9) system shows potential for diagnosing and treating conditions like myotonic dystrophy and ALS.
Area of Science:
- Genetics
- Molecular Biology
- Biotechnology
Background:
- Microsatellite repeat expansions in DNA lead to pathogenic RNA species.
- These toxic RNAs cause severe dominantly inherited neurological disorders, including myotonic dystrophy (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS).
- Effective diagnostic and therapeutic strategies targeting these repetitive RNAs are urgently needed.
Purpose of the Study:
- To develop a programmable CRISPR system for specifically visualizing and eliminating toxic repeat expansion RNAs.
- To evaluate the efficacy of this RNA-targeting CRISPR system in disease models and patient cells.
Main Methods:
- Development of a programmable CRISPR system, termed RNA-targeting Cas9 (RCas9).
- Testing RCas9 for specific targeting and elimination of microsatellite repeat expansion RNAs in both exogenous expression systems and patient-derived cells.
- Assessment of RCas9's ability to reverse disease-specific molecular and cellular phenotypes.
Main Results:
- RCas9 specifically targeted and efficiently eliminated toxic repeat expansion RNAs.
- Observed elimination of RNA foci across multiple disease models (DM1, DM2, C9-ALS, polyglutamine diseases).
- Demonstrated reduction of toxic protein products, restoration of protein localization, and reversal of splicing defects in patient cells.
Conclusions:
- The developed RCas9 system effectively targets and eliminates pathogenic RNAs responsible for repeat expansion disorders.
- RCas9 demonstrates significant potential for therapeutic applications in treating a range of debilitating genetic diseases.
- A truncated RCas9 variant suitable for adeno-associated viral vector delivery was successfully developed, enhancing therapeutic prospects.
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