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CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors.
J Vis Exp. 2019 09 14JV

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.

Authors+Show Affiliations

Medical College of Georgia, Augusta University.Medical College of Georgia, Augusta University.Medical College of Georgia, Augusta University.Medical College of Georgia, Augusta University.Medical College of Georgia, Augusta University; YAOTANG@augusta.edu.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Video-Audio Media

Language

eng

PubMed ID

31566614

Citation

Jin, Yue, et al. "CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors." Journal of Visualized Experiments : JoVE, 2019.
Jin Y, Shen Y, Su X, et al. CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors. J Vis Exp. 2019.
Jin, Y., Shen, Y., Su, X., Weintraub, N., & Tang, Y. (2019). CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors. Journal of Visualized Experiments : JoVE, (151). https://doi.org/10.3791/59432
Jin Y, et al. CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors. J Vis Exp. 2019 09 14;(151) PubMed PMID: 31566614.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors. AU - Jin,Yue, AU - Shen,Yan, AU - Su,Xuan, AU - Weintraub,Neal, AU - Tang,Yaoliang, Y1 - 2019/09/14/ PY - 2019/10/1/entrez PY - 2019/10/1/pubmed PY - 2020/6/25/medline JF - Journal of visualized experiments : JoVE JO - J Vis Exp IS - 151 N2 - 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. SN - 1940-087X UR - https://www.unboundmedicine.com/medline/citation/31566614/CRISPR/Cas9_Technology_in_Restoring_Dystrophin_Expression_in_iPSC_Derived_Muscle_Progenitors_ L2 - https://doi.org/10.3791/59432 DB - PRIME DP - Unbound Medicine ER -