Figure 7.

Restoration of DMD expression in transdifferentiated DMD EX45del myotubes

(A) Schematic illustration of CRISPR editing and transdifferentiation of edited cells into myotubes by overexpressing human MYOD transcription factor. Since the cells are primary fibroblasts that can only be passaged a few times without growth retardation, myotube reprogramming was performed for a pool of CRISPR-edited cells without pre-selection or enrichment of any indel types. (B) Examination of DMD mRNA expression by reverse transcription PCR in the transdifferentiated DMD Ex45del myotubes (day 12) edited by SpCas9 and dual gRNAs. Control is DMD Ex45del cells treated with SpCas9 only. Positive control: transdifferentiated normal human dermal fibroblasts (NHDFs). NTC, negative control for PCR reaction. (C) Representative immunofluorescence staining results for MHC (myosin heavy chain) and DMD (dystrophin protein) expression. MYOD expression was detected with dsRED signal. Nuclei are stained with DAPI. Magnification, 40×. Extended figures are shown in Figure S6.