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      Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing

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          CRISPR-mediated genome editing efficiently corrected cardiac abnormalities associated with DMD in human engineered heart tissue.


          Genome editing with CRISPR/Cas9 is a promising new approach for correcting or mitigating disease-causing mutations. Duchenne muscular dystrophy (DMD) is associated with lethal degeneration of cardiac and skeletal muscle caused by more than 3000 different mutations in the X-linked dystrophin gene ( DMD). Most of these mutations are clustered in “hotspots.” There is a fortuitous correspondence between the eukaryotic splice acceptor and splice donor sequences and the protospacer adjacent motif sequences that govern prokaryotic CRISPR/Cas9 target gene recognition and cleavage. Taking advantage of this correspondence, we screened for optimal guide RNAs capable of introducing insertion/deletion (indel) mutations by nonhomologous end joining that abolish conserved RNA splice sites in 12 exons that potentially allow skipping of the most common mutant or out-of-frame DMD exons within or nearby mutational hotspots. We refer to the correction of DMD mutations by exon skipping as myoediting. In proof-of-concept studies, we performed myoediting in representative induced pluripotent stem cells from multiple patients with large deletions, point mutations, or duplications within the DMD gene and efficiently restored dystrophin protein expression in derivative cardiomyocytes. In three-dimensional engineered heart muscle (EHM), myoediting of DMD mutations restored dystrophin expression and the corresponding mechanical force of contraction. Correcting only a subset of cardiomyocytes (30 to 50%) was sufficient to rescue the mutant EHM phenotype to near-normal control levels. We conclude that abolishing conserved RNA splicing acceptor/donor sites and directing the splicing machinery to skip mutant or out-of-frame exons through myoediting allow correction of the cardiac abnormalities associated with DMD by eliminating the underlying genetic basis of the disease.

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          Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects.

          Bacterial RNA-directed Cas9 endonuclease is a versatile tool for site-specific genome modification in eukaryotes. Co-microinjection of mouse embryos with Cas9 mRNA and single guide RNAs induces on-target and off-target mutations that are transmissible to offspring. However, Cas9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations.
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            Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy

            Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx 4cv mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.
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              Is Open Access

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

              CRISPR-Cpf1–mediated correction of Duchenne muscular dystrophy mutations in human cells and a mouse model.

                Author and article information

                Sci Adv
                Sci Adv
                Science Advances
                American Association for the Advancement of Science
                January 2018
                31 January 2018
                : 4
                : 1
                [1 ]Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
                [2 ]Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
                [3 ]Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
                [4 ]Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, NY 10016, USA.
                [5 ]Institute of Pharmacology and Toxicology, University Medical Center, Georg-August-University Göttingen, Göttingen, 37075, Germany.
                [6 ]DZHK (German Center for Cardiovascular Research), partner site Göttingen, Göttingen, Germany.
                [7 ]Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
                Author notes

                These authors contributed equally to this work.

                []Corresponding author. Email: eric.olson@ 123456utsouthwestern.edu (E.N.O.); chengzu.long@ 123456nyumc.org (C.L.)
                Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

                This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

                Funded by: doi http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: award349726
                Award ID: HL-130253, HL-077439, DK-099653, and AR-067294
                Funded by: doi http://dx.doi.org/10.13039/100000928, Welch Foundation;
                Award ID: award349727
                Award ID: 1-0025
                Funded by: doi http://dx.doi.org/10.13039/100005693, Parent Project Muscular Dystrophy;
                Award ID: award368827
                Funded by: German Research Foundation;
                Award ID: award368838
                Award ID: DFG ZI 708/7-1, 8-1, 10-1; SFB 937 TP18, SFB 1002 TPs B03, C04, S1; IRTG 1816 RP12
                Funded by: Robert A. Welch Foundation;
                Award ID: award368830
                Award ID: 1-0025
                Funded by: Foundation Leducq;
                Award ID: award368840
                Funded by: German Center for Cardiovascular Research;
                Award ID: award368833
                Funded by: Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center;
                Award ID: award368823
                Award ID: U54 HD-087351
                Funded by: German Federal Ministry for Science and Education;
                Award ID: award368835
                Award ID: BMBF FKZ 13GW0007A [CIRM-ET3]
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