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Cell. 2017 Aug 24;170(5):899-912.e10. doi: 10.1016/j.cell.2017.07.010. Epub 2017 Aug 10.

Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9.

Author information

1
Department 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.
2
Department of Molecular Genetics and Microbiology, Center for NeuroGenetics and the Genetics Institute, University of Florida, College of Medicine, Gainesville, FL, USA.
3
Department of Neurology, University of Florida, College of Medicine, Gainesville, FL, USA.
4
Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA; Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA, USA; Department of Chemistry, University of California, San Diego, La Jolla, CA, USA.
5
Department 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; Molecular Engineering Laboratory, A(∗)STAR, Singapore, Singapore; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. Electronic address: geneyeo@ucsd.edu.

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.

KEYWORDS:

ALS; CRISPR; Huntington’s disease; RNA-targeting Cas9; adeno-associated virus; gene therapy; microsatellite repeat expansion; minimal Cas9; myotonic dystrophy

PMID:
28803727
PMCID:
PMC5873302
DOI:
10.1016/j.cell.2017.07.010
[Indexed for MEDLINE]
Free PMC Article

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