Fig. 2 DMD iPSC-derived cardiomyocytes express dystrophin after Cpf1-mediated genome editing by reframing. DMD iPSC-derived cardiomyocytes express dystrophin.

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Fig. 2 DMD iPSC-derived cardiomyocytes express dystrophin after Cpf1-mediated genome editing by reframing. DMD iPSC-derived cardiomyocytes express dystrophin after Cpf1-mediated genome editing by reframing. (A) DMD skin fibroblast-derived iPSCs were edited by Cpf1 using gRNA (corrected DMD iPSCs) and then differentiated into cardiomyocytes (corrected cardiomyocytes) for analysis of genetic correction of the DMD mutation. (B) A DMD deletion of exons 48 to 50 results in splicing of exon 47 to 51, generating an out-of-frame mutation of dystrophin. Forward primer (F) targeting exon 47 and reverse primer (R) targeting exon 52 were used in RT-PCR to confirm the reframing strategy by Cpf1-meditated genome editing in cardiomyocytes. Uncorrected cardiomyocytes lack exons 48 to 50. In contrast, after reframing, exon 51 is placed back in frame with exon 47. (C) Sequencing of representative RT-PCR products shows that uncorrected DMD iPSC-derived cardiomyocytes have a premature stop codon in exon 51, which creates a nonsense mutation. After Cpf1-mediated reframing, the ORF of dystrophin is restored. Dashed red line denotes exon boundary. (D) Western blot analysis shows dystrophin expression in a mixture of DMD iPSC-derived cardiomyocytes edited by reframing with LbCpf1 or AsCpf1 and g1 gRNA. Even without clonal selection, Cpf1-mediated reframing is efficient and sufficient to restore dystrophin expression in the cardiomyocyte mixture. α-Myosin heavy chain (αMHC) is loading control. (E) Immunocytochemistry shows dystrophin expression in iPSC-derived cardiomyocyte (CM) mixtures following LbCpf1- or AsCpf1-mediated reframing. Red, dystrophin staining; green, troponin I staining. Scale bar, 100 μm. (F) Western blot analysis shows dystrophin expression in single clones (#2 and #5) of iPSC-derived cardiomyocytes following clonal selection after LbCpf1-mediated reframing. αMHC is loading control. (G) Immunocytochemistry shows dystrophin expression in clone #2 LbCpf1-edited iPSC-derived cardiomyocytes. Scale bar, 100 μm. (H) Quantification of mtDNA copy number in single clones (#2 and #5) of LbCpf1-edited iPSC-derived cardiomyocytes. Data are means ± SEM (n = 3). &P < 0.01 and #P < 0.005. ns, not significant. (I) Basal OCR of single clones (#2 and #5) of LbCpf1-edited iPSC-derived cardiomyocytes, and OCR in response to oligomycin, FCCP, rotenone, and antimycin A, normalized to cell number. Data are means ± SEM (n = 5). *P < 0.05, &P < 0.01, and #P < 0.005. Yu Zhang et al. Sci Adv 2017;3:e1602814 Copyright © 2017, The Authors