Journal: Molecular Therapy. Nucleic Acids

Article Title: Optimized genomic editing of a common Duchenne muscular dystrophy mutation in patient-derived muscle cells and a new humanized mouse model

doi: 10.1016/j.omtn.2025.102569

Figure Lengend Snippet: CRISPR-Cas9 single-cut strategy to correct a DMD Δ52 mutation (A) Schematic of DMD exons in which the shapes of the exons indicate whether splicing between adjacent exons retains the contiguous open reading frame (ORF). Exons 51–54 are highlighted as the primary region of focus in this study. (B) Deletion of exon 52 introduces a premature stop codon in exon 53, preventing dystrophin expression. Single-cut CRISPR-Cas9-mediated gene editing can either reframe exon 53 with INDELs or target the splice acceptor site to skip exon 53 and restore the ORF. (C) Human exon 53 DNA sequence with amino acid sequences of wild-type (WT) and Δ52 DMD protein. The red box indicates the premature stop codon created by the exon 52 deletion. The red arrowheads indicate the DNA DSB created by Sp Cas9-LRVQR. The hEx53sg13 sequence is underlined in the negative strand. The 5′-NGTG-3′ PAM sequence of Sp Cas9-LRVQR is highlighted in blue.

Article Snippet: Gels were run at 80 V for 30 min and switched to 130 V for 2 h, followed by a wet transfer to a polyvinylidene difluoride (PVDF) membrane at 100 V at 4°C for 90 min. For Cas9 protein detection, the PVDF membrane was blocked in blocking buffer (5% w/v nonfat dry milk, 1× Tris-buffered saline, and 0.1% Tween 20) at RT for 1 h and incubated with mouse anti-Cas9 primary antibody (MAC133, 1:1,000 Millipore Sigma) at 4°C overnight at 4°C overnight, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. For dystrophin protein detection, the PVDF membrane was incubated with mouse anti-dystrophin primary antibody (MANDYS8, 1:1,000; Sigma-Aldrich) at 4°C overnight, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. For vinculin protein detection, the PVDF membrane was incubated with mouse anti-vinculin primary antibody (V9131, 1:1,000; Sigma-Aldrich) at RT for 1 h, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. The PVDF membrane was developed by Western Blotting Luminol Reagent (Santa Cruz) according to manufacturer’s protocol and imaged by digital imager (Bio-Rad).

Techniques: CRISPR, Mutagenesis, Expressing, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Optimized genomic editing of a common Duchenne muscular dystrophy mutation in patient-derived muscle cells and a new humanized mouse model

doi: 10.1016/j.omtn.2025.102569

Figure Lengend Snippet: Restoration of dystrophin expression in DMD Δ52 patient-derived iPSC CMs using the Sp Cas9-LRVQR single-cut approach (A) Peripheral blood mononuclear cells (PBMCs) were collected from a Δ52 DMD patient and reprogrammed into DMD-iPSCs. The cells were edited using Sp Cas9-LRVQR and hEx53sg13. The corrected DMD-iPSCs were then differentiated into cardiomyocytes (CMs). (B) Dystrophin restoration in DMD Δ52 CMs following Sp Cas9-LRVQR-mediated single-cut gene editing as shown by immunofluorescence staining. Dystrophin is shown in red, troponin I in green, and DAPI in blue. Scale bars, 50 μm. (C) Western blot showing dystrophin protein restoration in corrected DMD Δ52 CMs after Sp Cas9-LRVQR single-cut gene editing. Vinculin is a loading control. (D) Genomic deep sequencing analysis of the on-target site. Blue bar shows editing efficiency of +1 bp insertion with Sp Cas9-LRVQR and hEx53sg13. Orange bar shows editing efficiency of +1 bp insertion without Sp Cas9-LRVQR and hEx53sg13. (E) Genomic deep sequencing analysis of the top eight predicted off-target sites associated with Sp Cas9-LRVQR and hEx53g13. OT refers to off-target. (F) RT-PCR of WT, Δ52, and corrected Δ52 CMs differentiated from patient-derived iPSCs (WT: 628 bp, Δ52: 416 bp, and corrected Δ52: 417 bp). The arrows indicated the positions of the forward and reversed primers for RT-PCR. (G) Percentage of +1 efficiency based on Sanger sequencing of cDNA H) Fragment of exon 53 cDNA sequence showing the +1 insertion of adenine after gene editing. The top sequence represents Δ52 CMs. The bottom sequence shows corrected Δ52 CMs. The red box and arrow show the insertion of adenine.

Article Snippet: Gels were run at 80 V for 30 min and switched to 130 V for 2 h, followed by a wet transfer to a polyvinylidene difluoride (PVDF) membrane at 100 V at 4°C for 90 min. For Cas9 protein detection, the PVDF membrane was blocked in blocking buffer (5% w/v nonfat dry milk, 1× Tris-buffered saline, and 0.1% Tween 20) at RT for 1 h and incubated with mouse anti-Cas9 primary antibody (MAC133, 1:1,000 Millipore Sigma) at 4°C overnight at 4°C overnight, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. For dystrophin protein detection, the PVDF membrane was incubated with mouse anti-dystrophin primary antibody (MANDYS8, 1:1,000; Sigma-Aldrich) at 4°C overnight, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. For vinculin protein detection, the PVDF membrane was incubated with mouse anti-vinculin primary antibody (V9131, 1:1,000; Sigma-Aldrich) at RT for 1 h, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. The PVDF membrane was developed by Western Blotting Luminol Reagent (Santa Cruz) according to manufacturer’s protocol and imaged by digital imager (Bio-Rad).

Techniques: Expressing, Derivative Assay, Immunofluorescence, Staining, Western Blot, Control, Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Therapy. Nucleic Acids

Article Title: Optimized genomic editing of a common Duchenne muscular dystrophy mutation in patient-derived muscle cells and a new humanized mouse model

doi: 10.1016/j.omtn.2025.102569

Figure Lengend Snippet: Intraperitoneal AAV9-mediated delivery of Sp Cas9-LRVQR and hEx53sg13 restores dystrophin expression in Δ52; h53 KI mice (A) Schematic representation of AAV9 vectors used to deliver Sp Cas9-LRVQR and hEx53sg13. Sp Cas9-LRVQR was inserted into a single-stranded AAV9 backbone, under the control of muscle-specific CK8e promoter. Two copies of the hEx53sg13 cassette were cloned into a double-stranded self-complementary AAV9 backbone (scAAV9), with expression driven by U6 and M11 RNA polymerase III promoters respectively. The stuffer sequence was included in the scAAV9 vector for optimal packaging. (B) Experimental timeline of systemic delivery of AAV9 via intraperitoneal (i.p.) injection in P4 mice at a total dose of 2E14 vg/kg. Tissues were analyzed three months after injection. (C) Immunofluorescence staining of heart, diaphragm, triceps, and TA showed lack of dystrophin expression in Δ52; h53 KI mice injected with saline and restoration of dystrophin expression in Δ52; h53 KI mice injected with AAV9-gene editing components. Dystrophin is shown in green, DAPI in blue. Scale bars, 50 μm (a single representative image is displayed, with a total of six AAV-injected mice, n = 6). Tissues are shown in transverse sections. (D) H&E staining of heart, diaphragm, triceps, and TA showed dystrophic muscle abnormalities including centralized nuclei, necrosis, and fibrosis in Δ52; h53 KI mice injected with saline, and restoration of muscle morphology in Δ52; h53 KI mice injected with AAV9-gene editing components. Scale bars, 200 μm. Tissues are shown in transverse sections.

Article Snippet: Gels were run at 80 V for 30 min and switched to 130 V for 2 h, followed by a wet transfer to a polyvinylidene difluoride (PVDF) membrane at 100 V at 4°C for 90 min. For Cas9 protein detection, the PVDF membrane was blocked in blocking buffer (5% w/v nonfat dry milk, 1× Tris-buffered saline, and 0.1% Tween 20) at RT for 1 h and incubated with mouse anti-Cas9 primary antibody (MAC133, 1:1,000 Millipore Sigma) at 4°C overnight at 4°C overnight, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. For dystrophin protein detection, the PVDF membrane was incubated with mouse anti-dystrophin primary antibody (MANDYS8, 1:1,000; Sigma-Aldrich) at 4°C overnight, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. For vinculin protein detection, the PVDF membrane was incubated with mouse anti-vinculin primary antibody (V9131, 1:1,000; Sigma-Aldrich) at RT for 1 h, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. The PVDF membrane was developed by Western Blotting Luminol Reagent (Santa Cruz) according to manufacturer’s protocol and imaged by digital imager (Bio-Rad).

Techniques: Expressing, Control, Clone Assay, Sequencing, Plasmid Preparation, Injection, Immunofluorescence, Staining, Saline

Journal: Molecular Therapy. Nucleic Acids

Article Title: Optimized genomic editing of a common Duchenne muscular dystrophy mutation in patient-derived muscle cells and a new humanized mouse model

doi: 10.1016/j.omtn.2025.102569

Figure Lengend Snippet: Intravenous AAV9-mediated delivery of Sp Cas9-LRVQR and hEx53sg13 restores dystrophin expression in Δ52; h53 KI mice (A) Experimental timeline of systemic delivery of AAV9 via facial vein injection in P2 mice at a total dose of 2E14 vg/kg. Tissues were analyzed three months after the injection. (B) Immunofluorescence staining of heart, diaphragm, triceps, and TA showed lack of dystrophin expression in Δ52; h53 KI mice injected with saline and restoration of dystrophin expression in Δ52; h53 KI mice injected with AAV9. Dystrophin is shown in green, DAPI in blue. Scale bars, 50 μm (a single representative image is displayed, with a total of four AAV9-injected mice, n = 4). Tissues are shown in transverse sections. (C) H&E staining of heart, diaphragm, triceps, and TA showed dystrophic muscle abnormalities including centralized nuclei, necrosis, and fibrosis in Δ52; h53 KI mice injected with saline, and restoration of muscle morphology in Δ52; h53 KI mice injected with AAV9. Scale bars, 200 μm. Tissues are shown in transverse sections.

Article Snippet: Gels were run at 80 V for 30 min and switched to 130 V for 2 h, followed by a wet transfer to a polyvinylidene difluoride (PVDF) membrane at 100 V at 4°C for 90 min. For Cas9 protein detection, the PVDF membrane was blocked in blocking buffer (5% w/v nonfat dry milk, 1× Tris-buffered saline, and 0.1% Tween 20) at RT for 1 h and incubated with mouse anti-Cas9 primary antibody (MAC133, 1:1,000 Millipore Sigma) at 4°C overnight at 4°C overnight, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. For dystrophin protein detection, the PVDF membrane was incubated with mouse anti-dystrophin primary antibody (MANDYS8, 1:1,000; Sigma-Aldrich) at 4°C overnight, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. For vinculin protein detection, the PVDF membrane was incubated with mouse anti-vinculin primary antibody (V9131, 1:1,000; Sigma-Aldrich) at RT for 1 h, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. The PVDF membrane was developed by Western Blotting Luminol Reagent (Santa Cruz) according to manufacturer’s protocol and imaged by digital imager (Bio-Rad).

Techniques: Expressing, Injection, Immunofluorescence, Staining, Saline

Journal: Molecular Therapy. Nucleic Acids

Article Title: Optimized genomic editing of a common Duchenne muscular dystrophy mutation in patient-derived muscle cells and a new humanized mouse model

doi: 10.1016/j.omtn.2025.102569

Figure Lengend Snippet: Restoration of dystrophin protein expression in humanized DMD mice following systemic delivery of Sp Cas9-LRVQR and hEx53sg13 (A) Western blot analysis shows dystrophin protein expression in various muscle groups of the Δ52; h53 KI mice injected with saline, AAV9 via FV, and AAV9 via i.p. Protein extracts from h53 KI control muscle groups were diluted and loaded as standards. Vinculin is used as the loading control ( n = 4 for FV and n = 6 for i.p.). The average percentage of dystrophin protein restoration is shown below the blot. Mouse numbers are shown above each line. (B) Quantification of dystrophin restoration in heart, diaphragm, triceps, and TA muscles, comparing two different administration routes: FV (orange) and i.p. (blue). Quantification of signals on western blots was done by ImageJ software. An unpaired t test with Welch’s correction was used to compare the injections efficiency. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001 ( n = 4 for FV and n = 6 for i.p.). (C) Quantification of percentage of +1 reframing efficiency by genomic DNA deep sequencing in heart, diaphragm, triceps, and TA muscles, comparing two different administration routes: FV (orange) and i.p. (blue). An unpaired t test with Welch’s correction was used to compare the injections efficiency. ∗∗ p < 0.01 and ∗∗∗ p < 0.001, ( n = 4 for FV and n = 6 for i.p.). (D) Forelimb grip-strength analysis conducted on h53 KI, and saline-treated and AAV9-injected Δ52; h53 KI mice at 3 months of age. The force measured in millinewtons (mN) was normalized to mouse body weight in grams (g). Results are displayed as mean ± SEM. One-way ANOVA with Holm-Šídák multiple comparisons test. ∗∗∗∗ p < 0.0001 ( n = 3 for h53 KI, n = 6 for i.p. and saline injected, and n = 4 for FV). (E) Measurement of serum creatine kinase (CK) in h53 KI control mice, and saline-treated and AAV9-treated Δ52; h53 KI mice. Results are displayed as mean ± SEM. One-way ANOVA with Holm-Šídák multiple comparisons test. ∗∗∗ p < 0.001, ( n = 3 for h53 KI, n = 6 for i.p. and saline injected, and n = 4 for FV).

Article Snippet: Gels were run at 80 V for 30 min and switched to 130 V for 2 h, followed by a wet transfer to a polyvinylidene difluoride (PVDF) membrane at 100 V at 4°C for 90 min. For Cas9 protein detection, the PVDF membrane was blocked in blocking buffer (5% w/v nonfat dry milk, 1× Tris-buffered saline, and 0.1% Tween 20) at RT for 1 h and incubated with mouse anti-Cas9 primary antibody (MAC133, 1:1,000 Millipore Sigma) at 4°C overnight at 4°C overnight, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. For dystrophin protein detection, the PVDF membrane was incubated with mouse anti-dystrophin primary antibody (MANDYS8, 1:1,000; Sigma-Aldrich) at 4°C overnight, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. For vinculin protein detection, the PVDF membrane was incubated with mouse anti-vinculin primary antibody (V9131, 1:1,000; Sigma-Aldrich) at RT for 1 h, followed by incubation with goat anti-mouse IgG (H + L)-HRP secondary antibody (1:10,000; Bio-Rad) at RT for 1 h. The PVDF membrane was developed by Western Blotting Luminol Reagent (Santa Cruz) according to manufacturer’s protocol and imaged by digital imager (Bio-Rad).

Techniques: Expressing, Western Blot, Injection, Saline, Control, Muscles, Software, Sequencing