title: A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells.
abstract: 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.
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Neurology, University of California, Los Angeles, CA 90095, USA.
authors.forename: Courtney S
authors.initials: CS
authors.lastname: Young
authors.affiliation: Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hicks
authors.affiliation: Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Department of Neurology, University of California, Los Angeles, CA 90095, USA.
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Ermolova
authors.affiliation: Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095, USA.
authors.forename: Haruko
authors.initials: H
authors.lastname: Nakano
authors.affiliation: Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA; CIRM Bridges Program, California State University, Northridge, CA 91330, USA.
authors.forename: Majib
authors.initials: M
authors.lastname: Jan
authors.affiliation: Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA; CIRM Bridges Program, California State University, Northridge, CA 91330, USA.
authors.forename: Shahab
authors.initials: S
authors.lastname: Younesi
authors.affiliation: Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
authors.forename: Saravanan
authors.initials: S
authors.lastname: Karumbayaram
authors.affiliation: Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Department of Neurology, University of California, Los Angeles, CA 90095, USA.
authors.forename: Chino
authors.initials: C
authors.lastname: Kumagai-Cresse
authors.affiliation: Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
authors.forename: Derek
authors.initials: D
authors.lastname: Wang
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
authors.forename: Jerome A
authors.initials: JA
authors.lastname: Zack
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
authors.forename: Donald B
authors.initials: DB
authors.lastname: Kohn
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095, USA.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nakano
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Human Genetics, University of California, Los Angeles, CA 90095, USA.
authors.forename: Stanley F
authors.initials: SF
authors.lastname: Nelson
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
authors.forename: M Carrie
authors.initials: MC
authors.lastname: Miceli
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Neurology, University of California, Los Angeles, CA 90095, USA. Electronic address: mspencer@mednet.ucla.edu.
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Spencer
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA. Electronic address: apyle@mednet.ucla.edu.
authors.forename: April D
authors.initials: AD
authors.lastname: Pyle
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D016189:Dystrophin / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D000072669:Gene Editing / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D016513:Mice, SCID; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics* / Q000378:metabolism / Q000473:pathology
chemical_list: D016189:Dystrophin