Correction of exon 2, exon 2-9 and exons 8-9 duplications in DMD patient myogenic cells by a single CRISPR/Cas9 system

Abstract

Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guide RNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2-9, and exons 8-9 in the DMD gene. Immunostaining on CRISPR-corrected derived myotubes demonstrated the rescue of dystrophin protein. Subsequent RNA sequencing of the DMD cells indicated rescue of genes of dystrophin related pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we further demonstrate the efficiency of a single guide CRISPR strategy capable of deleting multi-exon duplications in the DMD gene without significant off target effect. Our study contributes valuable insights into the safety and efficacy of using single guide CRISPR strategy as a potential therapeutic approach for DMD patients with duplications of variable size.

Figure 1

The single gRNA CRISPR strategy for correcting exon duplications in cells of DMD patients. (A) Results of the ddPCR quantification of the number of exons in DNA of the cell lines, with wild type immortalized myoblasts cell line C25 cells used as a control. (B) Scheme of the organization of the DMD gene in patient-derived immortalized myoblasts harboring exon duplications with location of the respective sgRNA. From top to bottom: exon 2 (Dup2), exons 2–9 (Dup2–9) and exon 8–9 (Dup8-9). (C) Relative cutting efficiency of the selected sgRNA for Dup2 and Dup2–9 assessed by CRISPR in-tube. (D) Relative cutting efficiency of the selected sgRNA for Dup8-9 assessed by TIDE analysis following transfection in HEK293 cells.

Figure 2

The single gRNA CRISPR strategy successfully corrects exon duplications in cells from DMD patients. (A) Results of the ddPCR for quantifying the number of exons in isolated clones following CRISPR editing. The clones where only one copy was detected are indicated in red. (B) Immunostaining of dystrophin by the antibody DYSB (in red) in the Dup2, Dup2–9, Dup8-9 (left panel) and Dup2 clone 7, Dup2–9 clone 14, Dup8-9 clone 12 (right panel) cell lines. Nuclei were stained with DAPI (in blue) The scale bar is 40 µm. (C) Capillary Western-blot analysis of dystrophin with the antibody DYSB and its quantification in the Dup2, Dup2–9, Dup8-9 and Dup2 clone 7, Dup2–9 clone 14, Dup8-9 clone 12 cell lines and C25 (positive control). The normalized signal corresponds to the ratio between the sample signal and the total protein signal of the same well. Three biological replicates were shown. A 2D representation of the capillary data is presented in Fig. S8B.

Figure 3

Correction of the DMD gene leads to its restored expression and the transcriptome profile of DMD disease. (A) Comparative level of dystrophin expression in the cell lines before and after gene editing as indicated by RNA-seq read counts. (B) The related exon counts of DMD. The DMD exons were divided into Beginning, Middle, and End exons. (C) Venn diagrams showing the overlap of modified genes in common between the different cell lines. The comparison between Dup2 and Dup2 clone 7 is in blue. between Dup2–9 and Dup2–9 clone 14 in yellow between Dup8-9 and Dup8-9 clone 12 in green. (D) Pathway networks of Gene Ontology (GO) analysis of the commonly dysregulated genes. Three clusters were identified: one related to actin filament, one related to actin filament, one related to response to potassium ion and another related to muscle function. (E) Heatmap visualization of the expression of the 43 commonly down-regulated and 21 up-regulated genes in the uncorrected and corrected cells.