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From gRNA Identification to the Restoration of Dystrophin Expression: A Dystrophin Gene Correction Strategy for Duchenne Muscular Dystrophy Mutations Using the CRISPR-Induced Deletion Method

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Duchenne Muscular Dystrophy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1687))

Abstract

The discovery of the CRISPR-Cas9 system raises hope for the treatment of many genetic disorders. We describe here an approach based on the use of a pair of single guide RNAs to form a hybrid exon that does not only restore the dystrophin gene reading frame but also results in the production of a dystrophin protein with an adequate structure of the central rod-domain, with a correct spectrin-like repeat. The therapeutic approach described here involved DMD patient cells having a deletion of exons 51–53 of the DMD gene.

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References

  1. Emery AE (2002) The muscular dystrophies. Lancet 359(9307):687–695

    Article  CAS  PubMed  Google Scholar 

  2. Emery AE (1991) Population frequencies of inherited neuromuscular diseases--a world survey. Neuromuscul Disord 1(1):19–29

    Article  CAS  PubMed  Google Scholar 

  3. Muntoni F, Torelli S, Ferlini A (2003) Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet Neurol 2(12):731–740

    Article  CAS  PubMed  Google Scholar 

  4. Bladen CL et al (2015) The TREAT-NMD DMD global database: analysis of more than 7,000 Duchenne muscular dystrophy mutations. Hum Mutat 36(4):395–402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Rybakova IN, Patel JR, Ervasti JM (2000) The dystrophin complex forms a mechanically strong link between the sarcolemma and costameric actin. J Cell Biol 150(5):1209–1214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Le Rumeur E (2015) Dystrophin and the two related genetic diseases, Duchenne and Becker muscular dystrophies. Bosn J Basic Med Sci 15(3):14–20

    Article  PubMed  PubMed Central  Google Scholar 

  7. Nicolas A et al (2015) Becker muscular dystrophy severity is linked to the structure of dystrophin. Hum Mol Genet 24(5):1267–1279

    Article  CAS  PubMed  Google Scholar 

  8. Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096

    Article  PubMed  Google Scholar 

  9. Jinek M et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821

    Article  CAS  PubMed  Google Scholar 

  10. Cong L et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Iyombe-Engembe JP et al (2016) Efficient restoration of the dystrophin gene reading frame and protein structure in DMD myoblasts using the CinDel method. Mol Ther Nucleic Acids 5:e283

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ran FA et al (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Centre de Recherche du CHU de Québec-Université Laval, 2705 Boul. Laurier, Québec, QC, Canada, G1V 4G2

    Benjamin Duchêne, Jean-Paul Iyombe-Engembe, Joël Rousseau, Jacques P. Tremblay & Dominique L. Ouellet

Authors
  1. Benjamin Duchêne
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  2. Jean-Paul Iyombe-Engembe
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  3. Joël Rousseau
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  4. Jacques P. Tremblay
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  5. Dominique L. Ouellet
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Corresponding author

Correspondence to Dominique L. Ouellet .

Editor information

Editors and Affiliations

  1. Istituto di Anatomia Umana e Biologia Cellulare, Università Cattolica del Sacro Cuore, Roma, Italy

    Camilla Bernardini

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Duchêne, B., Iyombe-Engembe, JP., Rousseau, J., Tremblay, J.P., Ouellet, D.L. (2018). From gRNA Identification to the Restoration of Dystrophin Expression: A Dystrophin Gene Correction Strategy for Duchenne Muscular Dystrophy Mutations Using the CRISPR-Induced Deletion Method. In: Bernardini, C. (eds) Duchenne Muscular Dystrophy. Methods in Molecular Biology, vol 1687. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7374-3_19

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  • DOI: https://doi.org/10.1007/978-1-4939-7374-3_19

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7373-6

  • Online ISBN: 978-1-4939-7374-3

  • eBook Packages: Springer Protocols

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