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Mol Cell. 2017 Nov 2;68(3):479-490.e5. doi: 10.1016/j.molcel.2017.09.033. Epub 2017 Oct 19.

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9.

Author information

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: eric.t.wang@ufl.edu.

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.

KEYWORDS:

C9ORF72/ALS/FTD; CRISPR; Cas9; RNA polymerase II; RNA toxicity; amyotrophic lateral sclerosis; microsatellite repeat disease; myotonic dystrophy; transcription

PMID:
29056323
PMCID:
PMC6013302
DOI:
10.1016/j.molcel.2017.09.033
[Indexed for MEDLINE]
Free PMC Article

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