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

Yanlin Wang; Lei Hao; Hongcai Wang; Katherine Santostefano; Arjun Thapa; John Cleary; Hui Li; Xiuming Guo; Naohiro Terada; Tetsuo Ashizawa; Guangbin Xia

doi:10.1016/j.ymthe.2018.09.003

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

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

Authors

Yanlin Wang¹, Lei Hao², Hongcai Wang³, Katherine Santostefano⁴, Arjun Thapa⁵, John Cleary⁶, Hui Li⁷, Xiuming Guo⁸, Naohiro Terada⁴, Tetsuo Ashizawa⁹, Guangbin Xia¹⁰

Affiliations

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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

3' Untranslated Regions
CRISPR-Cas Systems / genetics
Cell Differentiation / genetics
Gene Editing / methods
Genetic Therapy / methods
HEK293 Cells
Humans
Muscle, Skeletal / growth & development
Myocytes, Cardiac / physiology
Myotonic Dystrophy / genetics*
Myotonic Dystrophy / pathology
Myotonic Dystrophy / therapy
Myotonin-Protein Kinase / genetics*
Neural Stem Cells / physiology*
Neurons / physiology
RNA 3' Polyadenylation Signals / genetics
RNA, Guide, CRISPR-Cas Systems
Transfection
Trinucleotide Repeat Expansion / genetics*

Substances

3' Untranslated Regions
DMPK protein, human
RNA, Guide, CRISPR-Cas Systems
Myotonin-Protein Kinase

Grants and funding

K08 AR064836/AR/NIAMS NIH HHS/United States