Research ArticleMuscular Dystrophy

Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

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Science Translational Medicine  29 Nov 2017:
Vol. 9, Issue 418, eaan8081
DOI: 10.1126/scitranslmed.aan8081
  • Fig. 1. Generation of the ΔEx50 mouse model.

    (A) Strategy showing clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)–mediated genome editing approach to generate ΔEx50 mice. (B) Reverse transcription polymerase chain reaction (RT-PCR) analysis of muscle RNA using primers in exons 48 and 55 to validate deletion of exon 50 (ΔEx50). Bands for wild-type (WT) and ΔEx50 mouse dystrophin are 1045 and 936 base pair (bp), respectively. (C) RT-PCR products from ΔEx50 mouse muscle were sequenced to validate exon 50 deletion and generation of an out-of-frame sequence. (D) Hematoxylin and eosin (H&E) staining and immunostaining of dystrophin in the cardiac and skeletal muscles from WT (upper) and ΔEx50 (lower) mice at 2 months of age. (E) Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in the tibialis anterior and heart muscle from 2-month-old ΔEx50 mice. VCL expression in the skeletal muscle displays two isoforms compared to the cardiac muscle. (F) Serum creatine kinase (CK), a marker of muscle damage and membrane leakage, in WT, ΔEx50, and mdx mice at 2 months of age. n = 5. Data are represented as means ± SEM. Red arrow indicates dystrophin protein. Scale bars, 50 μm.

  • Fig. 2. Strategy for CRISPR/Cas9-mediated genome editing in ΔEx50 mice.

    (A) Scheme showing the CRISPR/Cas9-mediated genome editing approach to correct the reading frame in ΔEx50 mice by skipping exon 51. Gray exons are out of frame. (B) Illustration of single guide RNA (sgRNA) binding position and sequence for sgRNA-ex51. Protospacer adjacent motif (PAM) sequence for sgRNA is indicated in red. Black arrow indicates the cleavage site. Fw, foward primer; Rv, reverse primer. (C) Genomic deep-sequencing analysis of PCR amplicons generated across the exon 51 target site in 10T1/2 cells. Sequence of representative indels aligned with sgRNA sequence (indicated in blue) revealing insertions (highlighted in green) and deletions (highlighted in red). The line indicates the predicted exonic splicing enhancer (ESE) sequences located at the site of sgRNA. Black arrowhead indicates the cleavage site. (D) The muscle creatine kinase 8 (CK8e) promoter was used to express SpCas9. The U6, H1, and 7SK promoters for RNA polymerase III were used to express sgRNAs. GFP, green fluorescent protein.

  • Fig. 3. RT-PCR analysis of correction of reading frame.

    (A) RT-PCR of RNA from the tibialis anterior muscles of WT and ΔEx50 mice 3 weeks after intramuscular injection of the AAV9-sgRNA-51 and AAV9-Cas9 expression vectors. Lower dystrophin bands indicate deletion of exon 51. Primer positions in exons 48 and 53 are indicated. (B) Percentage of events detected at exon 51 after AAV9-Cas9/sgRNA-51 treatment using RT-PCR sequence analysis of TOPO-TA (topoisomerase-based thymidine to adenosine) generated clones. For each of four different samples, we generated and sequenced 40 clones. RT-PCR products were divided into four groups: Not edited (NE), exon 51–skipped (SK), reframed (RF), and out of frame (OF). (C) Sequence of the RT-PCR products of the ΔEx50-51 mouse dystrophin lower band confirmed that exon 49 spliced directly to exon 52, excluding exon 51. Sequence of RT-PCR products of ΔEx50 reframed (ΔEx50-RF) is also shown. (D) Deep-sequencing analysis of RT-PCR products from the upper band containing ΔEx50-NE and ΔEx50-RF was shown. Sequence of RT-PCR products revealing insertions (highlighted in green) is also depicted. n = 4. Data are represented as means ± SEM.

  • Fig. 4. Intramuscular injection of AAV9-Cas9/sgRNA-51 corrects dystrophin expression.

    (A) Tibialis anterior muscles of ΔEx50 mice were injected with AAV9 vector encoding sgRNA-51 and Cas9 (see Fig. 2) and were analyzed 3 weeks later by immunostaining for dystrophin. WT control (WT-CTL) mice and ΔEx50 control mice (ΔEx50-CTL) were injected with AAV9-Cas9 alone without sgRNAs. Indicated are percentages of dystrophin-positive myofibers in ΔEx50 mice receiving intramuscular injections of AAV9-Cas9/sgRNA-51 (ΔEx50-AAV9-sgRNA-51) compared to WT-CTL. (B) H&E staining of tibialis anterior muscles. (C) Western blot analysis of dystrophin (DMD) and VCL expression in tibialis anterior muscles 3 weeks after intramuscular injection of AAV9-Cas9 control or AAV9-Cas9/sgRNA-51. (D) Quantification of dystrophin expression from Western blots after normalization to VCL. Asterisk indicates nonspecific immunoreactive bands. n = 5 for AAV9-sgRNA-51. Scale bars, 50 μm.

  • Fig. 5. Systemic delivery of AAV9-Cas9/sgRNA-51 to ΔEx50 mice rescues dystrophin expression.

    Immunostaining for dystrophin in tibialis anterior, triceps, diaphragm, and cardiac muscles of ΔEx50 mice is shown. Immunostaining was performed 4 weeks after systemic injection of AAV9-Cas9 only (WT-CTL and ΔEx50-CTL) or AAV9-Cas9/sgRNA-51 (ΔEx50-AAV9-sgRNA-51). n = 5 for each group. Scale bars, 50 μm.

  • Fig. 6. Histological and functional analysis of dystrophin correction after systemic delivery of AAV9-Cas9/sgRNA-51 to ΔEx50 mice.

    (A) Western blot analysis of dystrophin (DMD) and VCL expression in tibialis anterior (TA) muscle, triceps, diaphragm, and cardiac muscle of ΔEx50 mice 4 weeks after systemic delivery of AAV9-Cas9 or AAV9-Cas9/sgRNA-51. (B) H&E staining of the TA muscle, triceps, and diaphragm muscle of ΔEx50 mice 4 weeks after systemic delivery of AAV9-Cas9 or AAV9-Cas9-sgRNA-51. (C) WT-CTL mice, ΔEx50 control mice, and ΔEx50 mice treated with AAV9-Cas9/sgRNA-51 (ΔEx50-AAV9-sgRNA-51) were subjected to grip strength testing to measure muscle performance (grams of force) that was normalized by body weight (BW). (D) Serum CK was measured in WT-CTL, ΔEx50-CTL, and ΔEx50-AAV9-sgRNA-51 mice. n = 5. Asterisk indicates nonspecific immunoreactive bands. Data are represented as means ± SEM. Significant differences between conditions are indicated by asterisk. ***P < 0.0005 using unpaired two-tailed Student’s t tests. Scale bars, 50 μm.

  • Fig. 7. Systemic delivery of AAV9-Cas9/sgRNA-51 to ΔEx50 mice rescues dystroglycan complex protein expression.

    Immunohistochemical staining for α-sarcoglycan and β-dystroglycan in tibialis anterior (TA) and triceps muscles 4 weeks after systemic injection of ΔEx50 mice with AAV9-Cas9 only (WT-CTL and ΔEx50-CTL) or AAV9-Cas9/sgRNA-51 (ΔEx50-AAV9-sgRNA-51). n = 5. Scale bars, 50 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/418/eaan8081/DC1

    Materials and Methods

    Fig. S1. ΔEx50 mouse model analyses.

    Fig. S2. Comparison of dystrophin staining in ΔEx50 and mdx mouse models at 1 month of age.

    Fig. S3. Characterization of ΔEx50 mice at different ages.

    Fig. S4. ESEs of exon 51.

    Fig. S5. Validation of sgRNAs in mouse 10T1/2 and human 293T cells.

    Fig. S6. Cas9 expression in injected muscles.

    Fig. S7. In vivo Dmd gene editing.

    Fig. S8. Off-target analyses for sgRNA-51.

    Fig. S9. Off-target sequence analyses for sgRNA-51.

    Fig. S10. Amplicon PCR deep-sequencing analyses for sgRNA-51.

    Fig. S11. Rescue of dystrophin expression after intramuscular injection of AAV9-Cas9 and AAV9-sgRNA-51 in the ΔEx50 mouse model.

    Fig. S12. AAV9-Cas9– and AAV9-sgRNA-51–injected muscle shows histological improvement after 3 weeks.

    Fig. S13. Quantification of histological improvement of AAV9-Cas9– and AAV9-sgRNA-51–injected muscle from ΔEx50 mice after 3 weeks.

    Fig. S14. Intramuscular injection of AAV9-Cas9 and AAV9-sgRNA-51 in ΔEx50 mice corrects dystrophin expression in the heart.

    Fig. S15. AAV9-Cas9 expression after systemic delivery in mice.

    Fig. S16. Dmd gene editing 4 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in mice.

    Fig. S17. Rescue of dystrophin expression 8 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in ΔEx50 mice.

    Fig. S18. Histological analysis of dystrophin correction 8 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in ΔEx50 mice.

    Fig. S19. Histological improvement of ΔEx50 mice 4 weeks after systemic injection of AAV9-Cas9 and AAV9-sgRNA-51.

    Table S1. Sequences of potential exonic off-target (OT) sites in the mouse genome.

    Table S2. Sequences of top 45 off-target (OT) sites in the mouse genome.

    Table S3. Primer sequences.

    References (47, 48)

  • Supplementary Material for:

    Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

    Leonela Amoasii, Chengzu Long, Hui Li, Alex A. Mireault, John M. Shelton, Efrain Sanchez-Ortiz, John R. McAnally, Samadrita Bhattacharyya, Florian Schmidt, Dirk Grimm, Stephen D. Hauschka, Rhonda Bassel-Duby, Eric N. Olson*

    *Corresponding author. Email: eric.olson{at}utsouthwestern.edu

    Published 29 November 2017, Sci. Transl. Med. 9, eaan8081 (2017)
    DOI: 10.1126/scitranslmed.aan8081

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. ΔEx50 mouse model analyses.
    • Fig. S2. Comparison of dystrophin staining in ΔEx50 and mdx mouse models at 1 month of age.
    • Fig. S3. Characterization of ΔEx50 mice at different ages.
    • Fig. S4. ESEs of exon 51.
    • Fig. S5. Validation of sgRNAs in mouse 10T1/2 and human 293T cells.
    • Fig. S6. Cas9 expression in injected muscles.
    • Fig. S7. In vivo Dmd gene editing.
    • Fig. S8. Off-target analyses for sgRNA-51.
    • Fig. S9. Off-target sequence analyses for sgRNA-51.
    • Fig. S10. Amplicon PCR deep-sequencing analyses for sgRNA-51.
    • Fig. S11. Rescue of dystrophin expression after intramuscular injection of AAV9-Cas9 and AAV9-sgRNA-51 in the ΔEx50 mouse model.
    • Fig. S12. AAV9-Cas9– and AAV9-sgRNA-51–injected muscle shows histological improvement after 3 weeks.
    • Fig. S13. Quantification of histological improvement of AAV9-Cas9– and AAV9-sgRNA-51–injected muscle from ΔEx50 mice after 3 weeks.
    • Fig. S14. Intramuscular injection of AAV9-Cas9 and AAV9-sgRNA-51 in ΔEx50 mice corrects dystrophin expression in the heart.
    • Fig. S15. AAV9-Cas9 expression after systemic delivery in mice.
    • Fig. S16. Dmd gene editing 4 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in mice.
    • Fig. S17. Rescue of dystrophin expression 8 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in ΔEx50 mice.
    • Fig. S18. Histological analysis of dystrophin correction 8 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in ΔEx50 mice.
    • Fig. S19. Histological improvement of ΔEx50 mice 4 weeks after systemic injection of AAV9-Cas9 and AAV9-sgRNA-51.
    • Table S1. Sequences of potential exonic off-target (OT) sites in the mouse genome.
    • Table S2. Sequences of top 45 off-target (OT) sites in the mouse genome.
    • Table S3. Primer sequences.
    • References (47, 48)

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