CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors

Yue Jin, Yan Shen, Xuan Su, Neal Weintraub, Yaoliang Tang

Research output: Contribution to journalArticle

Abstract

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the exhaustion of muscle progenitor cells. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene editing has the potential to restore the expression of the dystrophin gene. Autologous induced pluripotent stem cells (iPSCs)-derived muscle progenitor cells (MPC) can replenish the stem/progenitor cell pool, repair damage, and prevent further complications in DMD without causing an immune response. In this study, we introduce a combination of CRISPR/Cas9 and non-integrated iPSC technologies to obtain muscle progenitors with recovered dystrophin protein expression. Briefly, we use a non-integrating Sendai vector to establish an iPSC line from dermal fibroblasts of Dmdmdx mice. We then use the CRISPR/Cas9 deletion strategy to restore dystrophin expression through a non-homologous end joining of the reframed dystrophin gene. After PCR validation of exon23 depletion in three colonies from 94 picked iPSC colonies, we differentiate iPSC into MPC by doxycycline (Dox)-induced expression of MyoD, a key transcription factor playing a significant role in regulating muscle differentiation. Our results show the feasibility of using CRISPR/Cas9 deletion strategy to restore dystrophin expression in iPSC-derived MPC, which has significant potential for developing future therapies for the treatment of DMD.

Original languageEnglish (US)
JournalJournal of visualized experiments : JoVE
Issue number151
DOIs
StatePublished - Sep 14 2019

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Induced Pluripotent Stem Cells
Dystrophin
Stem cells
Muscle
Technology
Muscles
Stem Cells
Muscle Cells
Duchenne Muscular Dystrophy
Genes
Transcription factors
Fibroblasts
Doxycycline
Joining
Repair
Transcription Factors
Proteins
Gene Expression
Cell Line

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors. / Jin, Yue; Shen, Yan; Su, Xuan; Weintraub, Neal; Tang, Yaoliang.

In: Journal of visualized experiments : JoVE, No. 151, 14.09.2019.

Research output: Contribution to journalArticle

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