title: Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy.
abstract: The CRISPR/Cas9 genome-editing platform is a promising technology to correct the genetic basis of hereditary diseases. The versatility, efficiency and multiplexing capabilities of the CRISPR/Cas9 system enable a variety of otherwise challenging gene correction strategies. Here, we use the CRISPR/Cas9 system to restore the expression of the dystrophin gene in cells carrying dystrophin mutations that cause Duchenne muscular dystrophy (DMD). We design single or multiplexed sgRNAs to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45-55 and introducing shifts within exons or deleting one or more exons. Following gene editing in DMD patient myoblasts, dystrophin expression is restored in vitro. Human dystrophin is also detected in vivo after transplantation of genetically corrected patient cells into immunodeficient mice. Importantly, the unique multiplex gene-editing capabilities of the CRISPR/Cas9 system facilitate the generation of a single large deletion that can correct up to 62% of DMD mutations.
authors.affiliation: Department of Biomedical Engineering, Duke University, Room 136 Hudson Hall, Box 90281, Durham, North Carolina 27708, USA.
authors.forename: David G
authors.initials: DG
authors.lastname: Ousterout
authors.affiliation: Department of Biomedical Engineering, Duke University, Room 136 Hudson Hall, Box 90281, Durham, North Carolina 27708, USA.
authors.forename: Ami M
authors.initials: AM
authors.lastname: Kabadi
authors.affiliation: Department of Biomedical Engineering, Duke University, Room 136 Hudson Hall, Box 90281, Durham, North Carolina 27708, USA.
authors.forename: Pratiksha I
authors.initials: PI
authors.lastname: Thakore
authors.affiliation: Program in Computational Biology and Bioinformatics, Duke University, Durham, North Carolina 27708, USA.
authors.forename: William H
authors.initials: WH
authors.lastname: Majoros
authors.affiliation: 1] Center for Genomic and Computational Biology, Duke University, Durham, North Carolina 27708, USA [2] Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Timothy E
authors.identifier: 000000027629061X
authors.initials: TE
authors.lastname: Reddy
authors.affiliation: 1] Department of Biomedical Engineering, Duke University, Room 136 Hudson Hall, Box 90281, Durham, North Carolina 27708, USA [2] Center for Genomic and Computational Biology, Duke University, Durham, North Carolina 27708, USA [3] Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Gersbach
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002469:Cell Separation; D004195:Disease Models, Animal; D016189:Dystrophin / Q000235:genetics*; D005091:Exons; D005434:Flow Cytometry; D017353:Gene Deletion; D015316:Genetic Therapy / Q000379:methods; D016678:Genome; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D051379:Mice; D016513:Mice, SCID; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics*; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction
chemical_list: C484258:DMD protein, human; D016189:Dystrophin; C085339:apo-dystrophin 1