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. 2017 Sep 21;2(18):e95918. doi: 10.1172/jci.insight.95918

Figure 2. Correcting ΔEx8-9 iDMD by exon deletion to restore dystrophin expression.

Figure 2

(A) Three strategies were used to correct ΔEx8-9 iDMD by CRISPR/Cas9-mediated genomic editing: (i) deleting exons 3–7 to generate ΔEx3-9, (ii) deleting exons 6–7 to generate ΔEx6-9, and (iii) deleting exons 7–11 to generate ΔEx7-11. Shape and color of boxes denoting DMD exons indicate reading frame and protein coding domains. Yellow designates actin binding domain-1 (ABD-1). Blue marks part of the central rod domain. Red lines indicate actin binding sites (ABS1, ABS2, and ABS3). Red exon indicates exon with stop codon. (B) Illustration showing deletion of exons 3–7 to generate ΔEx3-9. Sequences of gRNAs and their targeting sites within intron 2 (top) and intron 7 (bottom). gRNAs were designed to target 3′ region of intron 2 (gRNA-2) and 5′ region of intron 7 (gRNA-7). Arrowheads mark targeting site of gRNAs. PAM sites are highlighted in red. (C) PCR genotyping of control, ΔEx8-9 iDMD, and two clones of ΔEx3-9 induced pluripotent cell (iPSC) lines using primers upstream and downstream of the gRNA targeting sites (top) and within intron 2 flanking the gRNA-2 targeting site (bottom). Sequencing of PCR product of ΔEx3-9 validates splicing of intron 2 to intron 7. PCR primers are indicated by arrows. Arrowhead indicates gRNA targeting site. M denotes marker lane. (D) RT-PCR analysis of dystrophin mRNA expression in control, ΔEx8-9 iDMD, and two clones of ΔEx3-9 iPSC–derived cardiomyocytes. Forward primer targeting exon 1 and reverse primer targeting exon 10 were used. Sequencing confirmed splicing of exon 2 to exon 10 restoring the open reading frame. α-Actinin was used as loading control. (E) Western blot analysis showing dystrophin protein expression in iPSC-derived cardiomyocytes using anti-dystrophin antibody. Vinculin was used as loading control. n = 7 for control and ΔEx8-9 iDMD, n = 3 for ΔEx3-9 clone 1 and ΔEx3-9 clone 2 n = 7 for control and ΔEx8-9 iDMD, n = 3 for ΔEx3-9 clone 1 and ΔEx3-9 clone 2. (F) Immunocytochemistry representations of iPSC-derived cardiomyocytes with anti-dystrophin (red) and anti–troponin I (green). Nuclei are stained with Hoechst 33342 (blue). Scale bar: 50 μm. n = 4 for control and ΔEx8-9 iDMD, n = 2 for ΔEx3-9 clone 1, and n = 1 for ΔEx3-9 clone 2.