Multiplex CRISPR/Cas9-based Genome Editing for Correction of Dystrophin Mutations That Cause Duchenne Muscular Dystrophy

Nat Commun. 2015 Feb 18;6:6244. doi: 10.1038/ncomms7244.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Cell Separation
  • Disease Models, Animal
  • Dystrophin / genetics*
  • Exons
  • Flow Cytometry
  • Gene Deletion
  • Genetic Therapy / methods
  • Genome*
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Male
  • Mice
  • Mice, SCID
  • Muscular Dystrophy, Duchenne / genetics*
  • Mutation*
  • Plasmids / metabolism
  • Polymerase Chain Reaction

Substances

  • DMD protein, human
  • Dystrophin
  • apo-dystrophin 1

Associated data

  • SRA/SRP051210