Fig. 7

In vivo editing of human DMD gene restores dystrophin expression and localization in muscle fibers. (A) Immunofluorescence detection of human dystrophin (green signal in the sarcolemma, white arrow), human lamin A+C (green signal in the nucleus), and laminin (red) in the TA muscle of CRISPR-targeted PDX DMD mice; nuclei were counterstained with DAPI (blue). Mice without editing (unedited) served as the negative control. Scale bar, 100 μm. b Representative box plots of the therapeutic efficacy of the different gene-editing strategies in vivo as determined by the ratio of the number of dystrophin-positive fibers (dystrophin⁺) to that of lamin A+C-positive nuclei (LaminA+C⁺); ∆46–54/Cas9, ∆46–54/Cas12a, and ∆45–55/Cas9 showed higher efficacy than INDEL50/Cas9 (P < 0.05, n = 8). c Confirmation of the presence of human cells and reframing of mutant DMD in PDX DMD mice by PCR. ∆45–55 yielded an intron 44/intron 55 junction, and ∆46–54 yielded an intron 45/intron 54 junction. hmtDNA, human mitochondrial DNA. d β-Dystroglycan restoration in human muscle fibers treated with different gene-editing strategies. Human dystrophin and β-dystroglycan are visible as green and red signals, respectively; sections were stained with DAPI (blue) to identify nuclei and labeled with an antibody against human Lamin A+C (green or red) to identify human nuclei. Mice without editing (unedited) served as a negative control. Scale bar, 50 μm