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. 2021 Apr 30;7(18):eabg4910. doi: 10.1126/sciadv.abg4910

Fig. 4. Base editing–mediated exon skipping restores dystrophin expression in human ∆Ex51 iPSC–derived cardiomyocytes.

Fig. 4

(A) Gene editing strategy to restore the in-frame ORF by exon skipping using base editing. (B) The hEx50 sgRNA-1 binding position in the region of the SDS of human DMD exon 50 (green). Sequence shows sgRNA (blue) and PAM (red). (C) Percentages of DNA editing of adenines in the editing window of ABEmax-SpCas9 with hEx50 sgRNA-1 following nucleofection in human ∆Ex51 iPSCs. On-target edit (A14) is colored green. Dots and bars represent results of different nucleofections and are means ± SEM (n = 3). (D) Representative Sanger sequencing chromatogram of the genomic region of the exon 50 SDS of human iPSCs following nucleofection with ABEmax-SpCas9 and hEx50 sgRNA-1. (E) RT-PCR analysis of RNA from single clones of healthy control (Ctrl), ∆Ex51, and corrected ∆Ex51 iPSC–derived cardiomyocytes after base editing. (F) Western blot analysis of dystrophin protein expression of Ctrl, ∆Ex51, and corrected ∆Ex51 iPSC–derived cardiomyocytes. Vinculin is the loading control. (G) Immunocytochemistry of dystrophin in Ctrl, ∆Ex51, and corrected ∆Ex51 iPSC–derived cardiomyocytes. Dystrophin is indicated in red. Cardiac troponin-I (TnI) is indicated in green. Nuclei are marked by DAPI (4′,6-diamidino-2-phenylindole) (blue). Scale bar, 50 μm.