CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice.

Zhang Y, Long C, Li H, McAnally JR, Baskin KK1,2,3, Shelton JM4, Bassel-Duby R, Olson EN,
Source: Sci Adv.
Publication Date: (2017)
Issue: 12, 3(4): 1-11
Research Area:
Stem Cells
Gene Expression
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
4D-Nucleofector® X-Unit
One hour before nucleofection, iPSCs were treated with 10 mMROCK inhibitor (Y-27632) and dissociated into single cells using Accutase (Innovative Cell Technologies Inc.). iPSCs (1 × 106) were mixed with 5 µg of the pLbCpf1-2A-GFP or pAsCpf1-2A-GFP plasmid and nucleofected using the P3 Primary Cell 4D-Nucleofector X Kit (Lonza) according to the manufacturer’s protocol. After nucleofection, iPSCs were cultured in mTeSR1 medium supplemented with 10 mM ROCK inhibitor, penicillin-streptomycin (1:100) (Thermo Fisher Scientific), and Primocin (100 mg/ml; InvivoGen). Three days after nucleofection, GFP(+) and GFP(-) cells were sorted by FACS and subjected to the T7E1 assay. Single clones derived from GFP(+) iPSCs were picked and sequenced. iPSCs were induced to differentiate into cardiomyocytes,
Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene (DMD), is characterized by fatal degeneration of striated muscles. Dilated cardiomyopathy is one of the most common lethal features of the disease. We deployed Cpf1, a unique class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effector, to correct DMD mutations in patient-derived induced pluripotent stem cells (iPSCs) and mdx mice, an animal model of DMD. Cpf1-mediated genomic editing of human iPSCs, either by skipping of an out-of-frame DMD exon or by correcting a nonsense mutation, restored dystrophin expression after differentiation to cardiomyocytes and enhanced contractile function. Similarly, pathophysiological hallmarks of muscular dystrophy were corrected in mdx mice following Cpf1-mediated germline editing. These findings are the first to show the efficiency of Cpf1-mediated correction of genetic mutations in human cells and an animal disease model and represent a significant step toward therapeutic translation of gene editing for correction of DMD