Fig. 7: SpCas9 mRNA reframes DYSF exon 44 and rescues dysferlin protein expression in MuSC from hEx44mut mice. | Nature Communications

Fig. 7: SpCas9 mRNA reframes DYSF exon 44 and rescues dysferlin protein expression in MuSC from hEx44mut mice.

From: Gene-editing in patient and humanized-mice primary muscle stem cells rescues dysferlin expression in dysferlin-deficient muscular dystrophy

Fig. 7

a Schematic overview of experiment setup. MuSC isolated from hind limbs of homozygous hEx44mut mice were transfected with SpCas9 mRNA plus sgRNA#3 or GFP mRNA. b Allele frequencies in unedited (n = 3) and edited (n = 6) MuSC from homozygous hEx44mut mice determined by NGS (mean ± SD). c Sanger-sequencing chromatogram of MuSC from homozygous hEx44mut mice transfected with SpCas9 mRNA plus sgRNA#3 compared to unedited cells. The protospacer and PAM sequences are underlined. The dotted vertical line indicates the expected DSB site. d Relative Dysf mRNA expression in edited and unedited MuSC from homozygous hEx44mut mice normalized to MuSC from homozygous hEx44wt mice (hEx44wt: n = 3; hEx44mut, unedited: n = 1, hEx44mut, edited: n = 2, mean ± SD). e Western blot analysis of dysferlin protein expression in edited and unedited MuSC from homozygous hEx44mut mice. MuSC from homozygous hEx44wt mice were used as control. f Quantification of dysferlin signal relative to α-tubulin from e using ImageJ. g Dysferlin immunostaining of reframed hEx44mut MuSC after differentiation into myotubes. Nuclei were stained with Hoechst. Scale bar: 20 μm. Source data are provided as a Source Data file.

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