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Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9.

Authors
  • Pinto, Belinda S1
  • Saxena, Tanvi1
  • Oliveira, Ruan1
  • Méndez-Gómez, Héctor R2
  • Cleary, John D1
  • Denes, Lance T1
  • McConnell, Ona1
  • Arboleda, Juan1
  • Xia, Guangbin3
  • Swanson, Maurice S1
  • Wang, Eric T4
  • 1 Department of Molecular Genetics & Microbiology, University of Florida, Gainesville, FL 32610, USA; Center for NeuroGenetics, University of Florida, Gainesville, FL 32610, USA.
  • 2 Department of Molecular Genetics & Microbiology, University of Florida, Gainesville, FL 32610, USA.
  • 3 Department of Neurology, University of Florida, Gainesville, FL 32610, USA; Center for NeuroGenetics, University of Florida, Gainesville, FL 32610, USA.
  • 4 Department of Molecular Genetics & Microbiology, University of Florida, Gainesville, FL 32610, USA; Center for NeuroGenetics, University of Florida, Gainesville, FL 32610, USA. Electronic address: [email protected]
Type
Published Article
Journal
Molecular cell
Publication Date
Nov 02, 2017
Volume
68
Issue
3
Identifiers
DOI: 10.1016/j.molcel.2017.09.033
PMID: 29056323
Source
Medline
Keywords
License
Unknown

Abstract

Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

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