Fig. 3: SpCas9 mRNA reframes DYSF exon 44 with >60% efficiency in MuSC from both patients with high precision and safety. | Nature Communications

Fig. 3: SpCas9 mRNA reframes DYSF exon 44 with >60% efficiency in MuSC from both patients with high precision and safety.

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

Fig. 3

a Schematic overview of experimental workflow. Patient and control MuSC were transfected with SpCas9 mRNA (3 µg per 150,000 cells), with or without sgRNA#3 (2 µg per 150,000 cells). b Sanger-sequencing chromatograms around the target site from patient MuSC transfected with or without sgRNA#3. The protospacer and PAM sequences are underlined in the upper unedited chromatogram. The dotted vertical line indicates the expected DSB site. c Allele frequencies in control and patient MuSC transfected with SpCas9 mRNA, with or without sgRNA#3, at day 4 and 9 post transfection determined by NGS (n = 3 replicates per donor and time point, mean ± SD). Data from the two control MuSC populations are plotted together. Ctrl: Control. d Frequency distribution of all indels in MuSC from the two patients at day 4 post-transfection with SpCas9 mRNA and sgRNA#3 (n = 3 replicates per patient, mean ± SD). e OTS identified by GUIDE-seq. The OTS sequences are aligned to the on-target site (top). The total number of GUIDE-seq reads aligned to each OTS from all samples, using both the GSP+ and GSP- primers, is plotted on the right. f All OTS identified by GUIDE-seq/CrispRGold (OTS 1-7), plus the top 10 and all exonic OTS predicted by CRISPOR (OTS 1, 3, 4 and OTS 8-21) for sgRNA#3 (Supplementary Table 3) were analyzed by NGS in patient MuSC collected 4–9 days after transfection with SpCas9 mRNA, with (n = 3) or without (n = 3 for OTS 2, 5, 6 and 7; n = 2 for all other OTS) sgRNA#3 (mean ± SD). Source data are provided as a Source Data file.

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