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 3

Correcting ΔEx8-9 iDMD by deleting exons 6 and 7 to restore dystrophin protein expression.

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Correcting ΔEx8-9 iDMD by deleting exons 6 and 7 to restore dystrophin p...
(A) Illustration showing deletion of exons 6–7 to generate ΔEx6-9. Sequences of guide RNAs (gRNAs) and their targeting sites within intron 5 (top) and intron 7 (bottom). gRNAs were designed to target the 3′ region of intron 5 (gRNA-5) and 5′ region of intron 7 (gRNA-7). Arrowheads mark targeting sites of gRNAs. Red exon indicates exon with stop codon. PAM sites are highlighted in red. (B) PCR genotyping of control, ΔEx8-9 iDMD, and two clones of ΔEx6-9 induced pluripotent stem cell (iPSC) lines using primers upstream and downstream of the gRNA targeting sites (top) and within intron 5 flanking the gRNA-5 targeting site (bottom). Sequencing of the PCR product of ΔEx6-9 validates splicing of intron 5 to intron 7. PCR primers are indicated by arrows. Arrowhead indicates gRNA targeting site. M denotes marker lane. (C) RT-PCR analysis of dystrophin mRNA expression in control, ΔEx8-9 iDMD, and two clones of ΔEx6-9 iPSC–derived cardiomyocytes. Forward primer targeting exon 5 and reverse primer targeting exon 10 were used. Sequencing confirmed splicing of exon 5 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 and ΔEx8-9 iDMD, n = 3 for ΔEx6-9 clone 1 and ΔEx6-9 clone 2. (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 and ΔEx8-9 iDMD, n = 3 for ΔEx6-9 clone 1 and ΔEx6-9 clone 2.