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Mol Ther. 2018 Nov 7;26(11):2617-2630. doi: 10.1016/j.ymthe.2018.09.003. Epub 2018 Sep 11.

Therapeutic Genome Editing for Myotonic Dystrophy Type 1 Using CRISPR/Cas9.

Author information

1
Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Henan 450000, China.
2
Department of Neurology, The Fifth People's Hospital of Chongqing, Chongqing 400062, China.
3
Department of Neurology, Affiliated Hospital of Binzhou Medical University, Binzhou City, Shandong Province, China; Department of Neurology, University of New Mexico, Albuquerque, NM, USA.
4
Department of Pathology, Immunology & Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL, USA.
5
Department of Neurology, University of New Mexico, Albuquerque, NM, USA.
6
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA.
7
Department of Neurology, University of Wisconsin, Madison, WI, USA.
8
Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
9
Houston Methodist Neurological Institute and Research Institute, 6670 Bertner Ave. R11-117, Houston, TX, USA.
10
Department of Neurology, University of New Mexico, Albuquerque, NM, USA; Department of Neuroscience, University of New Mexico, Albuquerque, NM, USA. Electronic address: guangbin.xia@gmail.com.

Abstract

Myotonic dystrophy type 1 (DM1) is caused by a CTG nucleotide repeat expansion within the 3' UTR of the Dystrophia Myotonica protein kinase gene. In this study, we explored therapeutic genome editing using CRISPR/Cas9 via targeted deletion of expanded CTG repeats and targeted insertion of polyadenylation signals in the 3' UTR upstream of the CTG repeats to eliminate toxic RNA CUG repeats. We found paired SpCas9 or SaCas9 guide RNA induced deletion of expanded CTG repeats. However, this approach incurred frequent inversion in both the mutant and normal alleles. In contrast, the insertion of polyadenylation signals in the 3' UTR upstream of the CTG repeats eliminated toxic RNA CUG repeats, which led to phenotype reversal in differentiated neural stem cells, forebrain neurons, cardiomyocytes, and skeletal muscle myofibers. We concluded that targeted insertion of polyadenylation signals in the 3' UTR is a viable approach to develop therapeutic genome editing for DM1.

KEYWORDS:

CRISPR/Cas9; DMPK; SaCas9; SpCas9 nickase; genome editing; induced pluripotent stem cell; myotonic dystrophy; nucleotide repeat expansion; polyadenylation; stem cell

PMID:
30274788
PMCID:
PMC6225032
[Available on 2019-11-07]
DOI:
10.1016/j.ymthe.2018.09.003
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