CRISPR/Cas9 Genome Editing in LGMD2A/R1 Patient-Derived Induced Pluripotent Stem and Skeletal Muscle Progenitor Cells

Figure 3

(a) Scheme displaying the new EXON3/4 CAPN3 conformation together with pyrograms emphasizing all silent introduced mutations TTT:TTC (Phe), CTG:CTT (Leu), EXON3 to EXON4 transition, as well as rescues in EXON3: 130Cys(TGC-LGMD2A) > Trp(TGG-WT) and EXON4: c.550delA. (b) CD82+ FACS sorting of skeletal muscle progenitor cells differentiated from LGMD2AiPSC: (i) unstained skeletal muscle population (left), (ii) differentiated and stained skeletal muscle cells with 43.85% gated myogenic progenitors/satellite-like cells (middle), and (iii) visualization of the shift of unstained and stained populations (red: unstained control; blue: CD82 stained cells) (right) and PAX7 immunohistochemistry of CD82+ progenitor cells after plating via cytospin. Pax7 (green) and DAPI (blue). Scale bar: 20 μm (right). (c) DNA sequencing electropherograms from genomic DNA of LGMD2A iPSC differentiated muscle cells at the exon3 390G > C CAPN3 locus (upper panel), at the exon4 550delA CAPN3 locus (middle panel), and after CRISPR/Cas9-gene-correction of the exon4 550delA CAPN3 locus (lower panel).

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