Research ArticleGENETICS

CRISPR-Cas9 corrects Duchenne muscular dystrophy exon 44 deletion mutations in mice and human cells

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Science Advances  06 Mar 2019:
Vol. 5, no. 3, eaav4324
DOI: 10.1126/sciadv.aav4324
  • Fig. 1 Exon 44–deleted DMD patient iPSC-derived cardiomyocytes express dystrophin after CRISPR-Cas9–mediated genome editing.

    (A) Schematic of the procedure for deriving and editing patient with DMD–derived iPSCs and iPSC-CMs. (B) Gene editing strategy for DMD exon 44 deletion. Deletion of exon 44 (black) results in splicing of exons 43 to 45, generating an out-of-frame stop mutation of dystrophin. Disruption of the splice junction of exon 43 or exon 45 results in splicing of exons 42 to 45 or exons 43 to 46, respectively, and restores the protein reading frame. The protein reading frame can also be restored by reframing exon 43 or 45 (green). (C) Sequence of sgRNAs targeting exon 43 splice acceptor and donor sites in the human DMD gene. The protospacer adjacent motif (PAM) (denoted as red nucleotides) of the sgRNAs is located near the exon 43 splice junctions. Exon sequence is represented by letters in bold uppercase. Intron sequence is represented by letters in lowercase. Arrowheads show sites of Cas9 DNA cutting with each sgRNA. Splice acceptor and donor sites are shaded in yellow. (D) Sequence of sgRNAs targeting exon 45 splice acceptor site in the human DMD gene. The PAM (denoted as red nucleotides) of the sgRNAs is located near the exon 45 splice acceptor site. The human and mouse conserved sequence is shaded in light blue. Exon sequence is represented by letters in bold uppercase. Intron sequence is represented by letters in lowercase. (E) Western blot analysis shows restoration of dystrophin expression in exon 43–edited (E43) and exon 45–edited (E45) ΔEx44 patient iPSC-CMs with sgRNAs (G) 3, 4, and 6, as indicated. Vinculin is the loading control. HC indicates iPSC-CMs from a healthy control. The second lane is the unedited ΔEx44 patient iPSC-CMs. (F) Immunostaining shows restoration of dystrophin expression in exon 43–edited and exon 45–edited ΔEx44 patient iPSC-CMs. Dystrophin is shown in red. Cardiac troponin I is shown in green. Nuclei are marked by 4′,6-diamidino-2-phenylindole (DAPI) stain in blue. Scale bar, 50 μm.

  • Fig. 2 Generation of mice with a DMD exon 44 deletion.

    (A) CRISPR-Cas9 editing strategy used for generation of mice with exon 44 deletion (ΔEx44). Exon 45 (red) is out of frame with exon 43. (B) RT-PCR analysis of TA muscles to validate deletion of exon 44. RT-PCR primers were in exons 43 and 46, and the amplicon size is 503 bp for WT mice and 355 bp for ΔEx44 DMD mice. RT-PCR products are schematized on the right (n = 3). (C) Sequencing of RT-PCR products from ΔEx44 DMD mouse muscle confirmed deletion of exon 44 and generation of a premature stop codon in exon 45, indicated by red asterisk. (D) Dystrophin staining of the TA, diaphragm, and heart of WT and ΔEx44 DMD mice. Dystrophin is shown in red. Nuclei are marked by DAPI stain in blue. Scale bar, 100 μm. (E) The Western blot analysis shows loss of dystrophin expression in the TA, gastrocnemius/plantaris (G/P) muscle, and heart of ΔEx44 mice. Vinculin is the loading control (n = 3). (F) H&E staining of the TA, diaphragm, and heart. Note extensive inflammatory infiltrate and centralized myonuclei in ΔEx44 sections. Inset boxes indicate areas of magnification shown below. Scale bars, 50 μm. (G) Serum creatine kinase (CK), a marker of muscle damage and membrane leakage, was measured in WT (C57BL/6 and C57BL/10), ΔEx44, and mdx mice. Data are represented as mean ± SEM. Unpaired Student’s t test was performed. *P < 0.005 (n = 6). (H) Representative trace of maximal tetanic force of EDL muscles in WT (blue) and ΔEx44 mice (red). P < 0.005 (n = 6). (I) Specific force of EDL muscles in WT (blue) and ΔEx44 mice (red). Data are represented as mean ± SEM. Unpaired Student’s t test was performed. **P < 0.001 (n = 6). (J) Forelimb grip strength analysis of WT and ΔEx44 mice. Data are represented as mean ± SEM. Unpaired Student’s t test was performed. **P < 0.001 (n = 6).

  • Fig. 3 Correction of Dmd exon 44 deletion in mice by intramuscular AAV9 delivery of gene editing components.

    (A) RT-PCR analysis of TA muscles from WT and ΔEx44 mice 3 weeks after intramuscular injection of gene editing components carried by AAV9. Lower dystrophin bands (179 bp) indicate skipping of exon 45. (B) Pie chart showing percentage of events detected at exon 45 after AAV-Cas9 and AAV-G6 treatment using RT-PCR sequence analysis of TOPO-TA generated clones. RT-PCR products were divided into four groups: NE, not edited; SK, exon 45 skipped; RF, reframed; and OF, out of frame. Data are represented as mean ± SEM (n = 3). (C) Sequences of RT-PCR products of WT, ΔEx44, and corrected ΔEx44 mice. In-frame sequences are shown in blue, including WT and exon 45–skipped sequences. Reframed sequence is shown in green, and out-of-frame sequence is shown in red. (D) Western blot analysis shows restoration of dystrophin expression in the TA muscle and heart of ΔEx44 mice. Vinculin is the loading control. (E) Quantification of the Western blot analysis in the TA muscle. Relative dystrophin intensity was calibrated with vinculin internal control. Data are represented as mean ± SEM. Unpaired Student’s t test was performed. *P < 0.005 (n = 3). (F) Immunostaining shows restoration of dystrophin in the TA muscle of ΔEx44 mice 3 weeks after intramuscular injection of gene editing components carried by AAV9. Dystrophin is shown in red. Nuclei are marked by DAPI stain in blue. Scale bar, 100 μm (n = 3). (G) H&E staining of the TA and heart in WT, ΔEx44, and corrected ΔEx44 mice. Inset boxes indicate areas of magnification shown below. Scale bars, 50 μm (n = 3).

  • Fig. 4 Systemic AAV9 delivery of gene editing components to ΔEx44 mice rescues dystrophin expression.

    (A) Immunostaining shows restoration of dystrophin in the TA, triceps, diaphragm, and heart of ΔEx44 mice 4 weeks after systemic delivery of AAV-Cas9 and AAV-G6 at the indicated ratios. Dystrophin is shown in red. Nuclei are marked by DAPI stain in blue. Scale bar, 100 μm (B) Western blot analysis shows restoration of dystrophin expression in the TA, triceps, diaphragm, and heart of ΔEx44 mice 4 weeks after systemic delivery of AAV-Cas9 and AAV-G6 at the indicated ratios. Vinculin the loading control (n = 4). (C) Maximal tetanic force of the EDL muscles in WT (blue), ΔEx44 DMD (red), and corrected ΔEx44 DMD (green) mice 4 weeks after systemic delivery of AAV-Cas9 and AAV-sgRNA at 1:5 and 1:10 ratios. P < 0.005 (n = 6). (D) Specific force (mN/mm2) of the EDL muscles in WT (blue), ΔEx44 DMD (red), and corrected ΔEx44 DMD (green) mice 4 weeks after systemic delivery of AAV-Cas9 and AAV-sgRNA at 1:5 and 1:10 ratios. Data are represented as mean ± SEM. One-way ANOVA was performed, followed by Newman-Keuls post hoc test. **P < 0.001 (n = 6).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/3/eaav4324/DC1

    Fig. S1. Analysis of sgRNAs that target the splice acceptor or donor sites for exons 43 and 45.

    Fig. S2. Characterization of the ΔEx44 mouse line.

    Fig. S3. Intramuscular AAV9 delivery of gene editing components rescues dystrophin expression.

    Fig. S4. Analysis of top 10 potential off-target sites.

    Fig. S5. Correction of ΔEx44 mice by systemic delivery of AAV9 expressing gene editing components.

    Fig. S6. Western blot analysis of corrected ΔEx44 mice by systemic delivery of AAV9 expressing gene editing components.

    Fig. S7. Histology of ΔEx44 mice after systemic delivery of AAV9 expressing gene editing components.

    Fig. S8. Quantification of histological improvement and qPCR analysis of corrected ΔEx44 DMD mice.

    Fig. S9. Histological analysis showing dystrophin restoration in the EDL muscle of corrected ΔEx44 DMD mice.

    Table S1. Primer sequences and media components.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Analysis of sgRNAs that target the splice acceptor or donor sites for exons 43 and 45.
    • Fig. S2. Characterization of the ΔEx44 mouse line.
    • Fig. S3. Intramuscular AAV9 delivery of gene editing components rescues dystrophin expression.
    • Fig. S4. Analysis of top 10 potential off-target sites.
    • Fig. S5. Correction of ΔEx44 mice by systemic delivery of AAV9 expressing gene editing components.
    • Fig. S6. Western blot analysis of corrected ΔEx44 mice by systemic delivery of AAV9 expressing gene editing components.
    • Fig. S7. Histology of ΔEx44 mice after systemic delivery of AAV9 expressing gene editing components.
    • Fig. S8. Quantification of histological improvement and qPCR analysis of corrected ΔEx44 DMD mice.
    • Fig. S9. Histological analysis showing dystrophin restoration in the EDL muscle of corrected ΔEx44 DMD mice.
    • Table S1. Primer sequences and media components.

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