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

Abstract

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.

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. (Δ) 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 mm.

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.