Prevention of early-onset cardiomyopathy in Dmd exon 52–54 deletion mice by CRISPR-Cas9-mediated exon skipping

Figures

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  Figure 1. In vivo removal of exon 55 using a dual guide strategy in Dmd Δ52–54 mice restores dystrophin expression in the heart (A) Schematic of sgRNAs, in54g2 and in55g4, designed to remove a region of 5.6 kb in Dmd Δ52–54 that encompasses exon 55. Δ52–54 mice carry an out-of-frame deletion (out-of-frame exons in orange), and removal of exon 55 will convert the Dmd Δ52–54 to the Dmd Δ52–55 in-frame deletion. (B) The two sgRNAs (blue and orange arrows) and SaCas9 were packaged into AAVs and injected systemically via the temporal vein. Mice were analyzed 6 weeks post injection. (C) RT-PCR of cDNA synthesized from gastrocnemius (G), triceps (T), diaphragm (D), and heart (H) tissues of mice treated with either GFP or the dual guide strategy. (D) Sanger sequencing of an edited amplicon from RT-PCR derived from heart tissue. (E) Immunofluorescence staining of heart tissue detected dystrophin-positive fibers after dual guide treatment. Scale bar, 100 μm. (F) Western blot of protein from heart tissues of GFP and dual-guide-treated mice (M1, M2, M3), probing for the presence of dystrophin (anti-dys), SaCas9 (anti-HA), and vinculin (anti-vinculin).

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  Figure 2. Single guide removal of exon 55 in Dmd Δ52–54 mice enhanced editing efficiency in the heart compared with the dual guide strategy (A) A single guide was designed to target and disrupt the exon 55 splice donor site (consensus sequence: AGgtragt; R: A or G) to enable exon 55 exclusion. (B) The plasmid expressing the in55g1_SD sgRNA (purple arrow) and SaCas9 was packaged into AAV9 and administered systemically through the temporal vein into Δ52–54 neonates. Treated mice were analyzed 6 weeks post injection. (C) RT-PCR of RNA derived from tibialis anterior (TA), triceps (Tri), heart (H), gastrocnemius (G), and diaphragm (D) tissues from GFP and single-guide-treated mice. (D) Immunofluorescence staining of heart and gastrocnemius tissues detected dystrophin-positive fibers in single-guide-treated mice. Scale bar, 100 μm. (E) Western blot probing for the presence of dystrophin (anti-dystrophin), SaCas9 (anti-HA), and vinculin (anti-vinculin) in GFP- and single-guide-treated mice. The percentage of restored dystrophin relative to wild-type (WT) levels for each treated sample is included in the lane’s label.

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  Figure 3. The high guide dosage approach does not significantly enhance dystrophin restoration in Dmd Δ52–54 mice (A) A plasmid packaging three additional copies of the in55g1_SD sgRNA was added to the single guide strategy, establishing the high guide dosage approach. Δ52-54 mice were injected systemically with the high guide dosage approach at P2 and analyzed at 12 weeks. (B) RT-PCR of cDNA of gastrocnemius (G), triceps (T), diaphragm (D), and heart (H) tissues of untreated mice and a high-guide-dosage-treated mouse. (C) The level of edited transcripts in triceps and heart tissues of GFP-, single-guide-, and high-guide-dosage-treated mice 12 weeks post injection were analyzed by qRT-PCR utilizing the expression ratio between the Dmd exon 51–56 junction and the WT Dmd transcript. (D) Immunofluorescence staining of gastrocnemius, triceps, diaphragm, and heart tissues from WT and high-guide-treated mice detected dystrophin-positive fibers. E) Immunofluorescence staining of heart tissues from WT and single-guide- and high-guide-dosage-treated mice detected dystrophin-positive fibers. Scale bar, 100 μm. Quantification demonstrated a significant increase in dystrophin-positive fibers in the…

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  Figure 4. High guide dosage treatment in Dmd Δ52–54 mice, preventing functional decline in the heart Echocardiography was used to analyze the hearts of WT and GFP- and high-guide-dosage-treated Δ52–54 mice to determine the (A) heart rate, (B) anterior ventricular wall thickness, and (C) fractional shortening. Statistical analyses were performed with Student’s t test. ∗p < 0.05, ∗∗p < 0.01.