Journal: bioRxiv

Article Title: CRISPR-Cas9 mediated endogenous utrophin upregulation improves Duchenne Muscular Dystrophy

doi: 10.1101/2023.04.18.536394

Figure Lengend Snippet: (A) Schematic representation of the timeline for generating 3D mini muscle starting from treated or untreated myoblasts. (B) TIDE quantification of the indels generated by the gRNA Let-7c-1 in 3D hDMD mini-muscle. Bars represent mean ± SEM of n = 3 per condition. Dots represent each experiment. (C) Indel profiles generated by the gRNA-hLet-7c-1 in 3D hDMD mini-muscle. In the inset are indicated the % at which each nucleotide is inserted in the +1 indels. Bars are mean ± SEM of n = 3 per condition. (D) Quantitative PCR analysis of utrophin A mRNA expression level, normalized with gapdh, after editing with the indicated gRNAs in 3D hDMD mini-muscle. Bars are mean ± SEM of n = 3 per condition. Dots represent each experiment. *p < 0.05. (E) Relative utrophin protein expression in 3D DMD muscles after editing with the indicated gRNA was determined by western blot and standardized for GAPDH loading. Bar graph: WB quantification. Bars represent mean ± SEM of n = 2 per condition. Dots represent each experiment. (F) Immunofluorescence staining for utrophin and myosin heavy chain (MyHC) on transverse sections of 3D DMD muscles show a higher utrophin signal after treatment with gRNA-hLet-7c compared to control. n = 4 per group. Scale bar: 100 µm. (G) Quantification of the immunostaining shown in (F) . Bars represent mean ± SEM of n = 3 per condition. Dots represent each experiment.

Article Snippet: Human DMD myoblasts were maintained in Smooth Muscle Cell Growth Medium (C-23060, PromoCell) supplemented with 20% fetal bovine serum gold (PAA) and 1% penicillin– streptomycin (Invitrogen).

Techniques: Generated, Real-time Polymerase Chain Reaction, Expressing, Muscles, Western Blot, Immunofluorescence, Staining, Control, Immunostaining