Fig. 2

Screening gRNAs for CRISPR/Cas9-mediated targeting of the DMD gene and reframing of mutant DMD in DMD–MDSCs. a Schematic illustration of gRNAs targeting introns 45 and 54 of the DMD gene (top row). Red and black arrows indicate the target sites of gRNAs and the primer binding sites for the PCR experiments shown in d. Illustration of a dual-fluorescence reporter plasmid (bottom row). b HEK293 cells were co-transfected with the reporter construct and Cas9 (top row) or Cas9/gRNA1–4 (bottom row) by electroporation, and EGFP and tdTomato (Td) expression was observed with a fluorescence microscope 2 days later. Scale bar, 100 μm. c Flow cytometry analysis of HEK293 cells co-transfected with the reporter construct and Cas9/gRNA (n = 2). d Confirmation of the reframing of mutant DMD in DMD–MDSCs by PCR and Sanger sequencing; ∆45–55 yielded an intron 44/intron 55 junction (left column), ∆46–54 yielded an intron 45/intron 54 junction (middle column), and INDEL50 yielded T7E1-cleaved PCR amplicon fragments of the expected sizes (red arrowheads); the red arrow indicates the gRNA target site (right column). M, marker; I, intron; E, exon. e Targeting efficiency of gRNAs in DMD-MDSCs. f The ratio of different types of indels in INDEL50. The numbers of reads of different indel types in three replicates was averaged, and the ratio was calculated. INSERT 3 N+2 and DELETE 3 N+1 were in-frame mutations