A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells.

Young CS, Hicks MR, Ermolova NV, Nakano H, Jan M, Younesi S, Karumbayaram S, Kumagai-Cresse C, Wang D, Zack JA, Kohn DB, Nakano A, Nelson SF, Miceli MC, Spencer MJ, Pyle AD.
Source: Cell Stem Cell
Publication Date: (2016)
Issue: 18(4): 533-40
Research Area:
Stem Cells
Gene Expression
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
4D-Nucleofector® X-Unit
In brief, hiPSCs were pre-treated with 10µM ROCK inhibitor Y-27632 (ROCKi, Tocris Bioscience) for 1hr and trysinized into a single cell suspension with TryplE (Life Technologies). 8x105 hiPSCs were nucleofected per 100µl cuvette using solution P3, 2µg or 3.5µg total DNA, and program CA-137 (Lonza). pMAX GFP (Lonza) was used as a transfection control. After nucleofection, cells were immediately plated in mTeSR1 with ROCKi. For selection, 0.35µg/ml of puromycin in mTeSR1 was added to the cells for 24hrs the day after nucleofection
Mutations in DMD disrupt the reading frame, prevent dystrophin translation, and cause Duchenne muscular dystrophy (DMD). Here we describe a CRISPR/Cas9 platform applicable to 60% of DMD patient mutations. We applied the platform to DMD-derived hiPSCs where successful deletion and non-homologous end joining of up to 725 kb reframed the DMD gene. This is the largest CRISPR/Cas9-mediated deletion shown to date in DMD. Use of hiPSCs allowed evaluation of dystrophin in disease-relevant cell types. Cardiomyocytes and skeletal muscle myotubes derived from reframed hiPSC clonal lines had restored dystrophin protein. The internally deleted dystrophin was functional as demonstrated by improved membrane integrity and restoration of the dystrophin glycoprotein complex in vitro and in vivo. Furthermore, miR31 was reduced upon reframing, similar to observations in Becker muscular dystrophy. This work demonstrates the feasibility of using a single CRISPR pair to correct the reading frame for the majority of DMD patients.