Functional correction of dystrophin actin binding domain mutations by genome editing
Viktoriia Kyrychenko, Sergii Kyrychenko, Malte Tiburcy, John M. Shelton, Chengzu Long, Jay W. Schneider, Wolfram-Hubertus Zimmermann, Rhonda Bassel-Duby, Eric N. Olson
Viktoriia Kyrychenko, Sergii Kyrychenko, Malte Tiburcy, John M. Shelton, Chengzu Long, Jay W. Schneider, Wolfram-Hubertus Zimmermann, Rhonda Bassel-Duby, Eric N. Olson
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Research Article Muscle biology

Functional correction of dystrophin actin binding domain mutations by genome editing

Abstract

Dystrophin maintains the integrity of striated muscles by linking the actin cytoskeleton with the cell membrane. Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD) that result in progressive, debilitating muscle weakness, cardiomyopathy, and a shortened lifespan. Mutations of dystrophin that disrupt the amino-terminal actin-binding domain 1 (ABD-1), encoded by exons 2–8, represent the second-most common cause of DMD. In the present study, we compared three different strategies for CRISPR/Cas9 genome editing to correct mutations in the ABD-1 region of the DMD gene by deleting exons 3–9, 6–9, or 7–11 in human induced pluripotent stem cells (iPSCs) and by assessing the function of iPSC-derived cardiomyocytes. All three exon deletion strategies enabled the expression of truncated dystrophin protein and restoration of cardiomyocyte contractility and calcium transients to varying degrees. We show that deletion of exons 3–9 by genomic editing provides an especially effective means of correcting disease-causing ABD-1 mutations. These findings represent an important step toward eventual correction of common DMD mutations and provide a means of rapidly assessing the expression and function of internally truncated forms of dystrophin-lacking portions of ABD-1.

Authors

Viktoriia Kyrychenko, Sergii Kyrychenko, Malte Tiburcy, John M. Shelton, Chengzu Long, Jay W. Schneider, Wolfram-Hubertus Zimmermann, Rhonda Bassel-Duby, Eric N. Olson

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Figure 6

Correction of DMD patient–derived iPSCs by deleting exons 8 and 9.

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Correction of DMD patient–derived iPSCs by deleting exons 8 and 9. (A) S...
(A) Strategy showing CRISPR/Cas9-mediated genomic editing of DMD (Duchene muscular dystrophy) patient (pΔEx3-7) to generate corrected pΔEx3-9 induced pluripotent cell (iPSC) line. Shape and color of boxes denoting DMD exons indicate reading frame and protein coding domains. Yellow designates ABD-1. Blue marks part of central rod domain. Red lines indicate ABS1. Arrowheads mark targeting sites of guide RNAs (gRNAs). Red box marks exon with stop codon. Sequences of gRNAs and their targeting sites within intron 7 (top) and intron 9 (bottom). gRNAs were designed to target the 3′ region of intron 7 (gRNA-7) and 5′ region of intron 9 (gRNA-9). PAM sites are highlighted in red. (B) PCR genotyping of pΔEx3-7and pΔEx3-9 iPSC lines using primers upstream and downstream of the gRNA targeting sites. Sequencing of PCR product of pΔEx3-9 validates splicing of intron 7 to intron 9. PCR primers are indicated by arrows. Arrowheads mark targeting sites of gRNAs. M denotes marker lane. (C) RT-PCR analysis of dystrophin mRNA expression in control, pΔEx3-7, and pΔEx3-9 iPSC–derived cardiomyocytes. Forward primer targeting 5′UTR and reverse primer targeting exon 10 were used. Sequencing of pΔEx3-7 confirmed splicing of exon 2 to exon 8, introducing a stop-codon. Sequencing pΔEx3-9 confirmed splicing of exon 2 to exon 10, restoring the open reading frame. α-Actinin was used as loading control. (D) 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, n = 3 for pΔEx3-7, and n = 3 for pΔEx3-9. (E) 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, n = 2 for pΔEx3-7, and n = 2 for pΔEx3-9. (F) Relative time to peak (TTP), (G) decay (τ), (H) transient duration (TD), and (I) percent of arrhythmic cells were measured based on calcium activity of control (n = 45), DMD patient pΔEx3-7 (n = 40), and corrected pΔEx3-9 (n = 65) iPSC–derived cardiomyocytes from 3–5 independent experiments. Data are represented as mean ± SEM. *P < 0.05 by one-way ANOVA (F–H).