Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA

Abstract

Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. DMD is characterized by progressive muscle weakness and a shortened life span, and there is no effective treatment. We used clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)-mediated genome editing to correct the dystrophin gene (Dmd) mutation in the germ line of mdx mice, a model for DMD, and then monitored muscle structure and function. Genome editing produced genetically mosaic animals containing 2 to 100% correction of the Dmd gene. The degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of gene correction, likely reflecting an advantage of the corrected cells and their contribution to regenerating muscle. With the anticipated technological advances that will facilitate genome editing of postnatal somatic cells, this strategy may one day allow correction of disease-causing mutations in the muscle tissue of patients with DMD.

Fig. 1. CRISPR/Cas9–mediated Dmd correction in mdx mice

(A) Schematic of the targeted exon of mouse Dmd and sequence from wild-type (upper) and mdx mice (lower).The mdx point mutation (C to T) is marked in red, and the premature stop codon is underlined. (B) Schematic of the 20-nucleotide sgRNA target sequence of the mdx allele (blue) and the PAM (red).The red arrowhead indicates the Cas9 cleavage site. ssODN, which contains 90 base pairs (bp) of homology sequence flanking each side of the target site, was used as HDR template. ssODN incorporates four silent mutations (green) and adds a TseI restriction enzyme site (underlined) for genotyping and quantification of HDR-mediated gene editing (fig. S1B). (C) Schematic for the gene correction by HDR or NHEJ.The corresponding DNA and protein sequences are shown in fig. S2A. (D) Strategy of the gene correction in mdx mice via germline gene therapy. (E) Genotyping results of mdx-C mice with mosaicism of 2 to 100% corrected Dmd gene. Undigested PCR product (upper panel),TseI digestion (middle panel), and T7E1 digestion (lower panel) on a 2% agarose gel. The red arrowhead in the middle panel marks the DNA band indicating HDR-mediated correction generated by TseI digestion. The blue arrowhead marks the DNA band of the uncorrected mdx allele.The relative intensity of the DNA bands (indicated by blue and red arrowheads) reflects the percentage of HDR in the genomic DNA. The percentage of HDR is located under the middle panel. The band intensity was quantified by ImageJ (NIH). The blue and red arrowheads in the lower panel indicate uncut and cut bands by T7E1, respectively. M denotes size marker lane. bp indicates the length of the marker bands.

Fig. 2. Histological analysis of muscles from wild-type, mdx, and mdx-C mice

Immunostaining and histological analysis of muscles from 7- to 9-week-old wild-type, mdx, and mdx-C mice (HDR-17%, HDR-41%, or NHEJ-83%). Dystrophin immunofluorescence (green) in wild-type mice is present in all muscles, including quadriceps, soleus,diaphragm, and heart, and is absent in mdx mice,except for a single revertant fiber in skeletal muscle. Skeletal muscle from the HDR-17% mouse has a characteristic pattern of clusters of dystrophin-positive fibers adjacent to clusters of dystrophin-negative fibers, whereas HDR-41% or NHEJ-83% mdx-C skeletal muscle is composed of dystrophin-positive myofibers only.White arrows indicate the adjacent clusters of dystrophin-positive fibers. Scale bar, 100 μm.

Fig. 3. Analysis of satellite cells from mdx-C mice and a model for rescue of muscular dystrophy by CRISPR/Cas9–mediated genomic correction

(A) Frozen sections of mdx-C gastrocnemius were mounted onto polyethylene membrane frame slides and immunohistochemically stained for Pax-7, a marker for satellite cells. Cross sections of muscle before (left) and after (right) laser dissection show the precise isolation of satellite cells (brown, in red circle). Scale bar, 25 μm. (B) PCR products corresponding to Dmd exon 23 were generated from genomic DNA isolated from satellite cells of mdx-C mice. PCR products were sequenced and show that CRISPR/Cas9–mediated genomic editing corrected a subset of satellite cells in vivo. Purple arrow indicates the corrected allele mediated by HDR. Green arrows indicate the silent mutation sites. The corresponding amino acid residues are shown under the DNA sequence.The red box indicates the corrected site. (C) A model for rescue of muscular dystrophy by CRISPR/Cas9–mediated genomic correction. There are three types of myo-fibers in mdx-C mice: (i) normal dystrophin-positive myofibers (green membrane) and satellite cells originating from corrected progenitors (green nuclei); (ii) dystrophic dystrophin-negative myofibers (brown membrane) and satellite cells originating from mdx progenitors (brown nuclei); and (iii) mosaic dystrophin-positive myofibers with centralized nuclei (green and brown nuclei) generated by fusion of corrected and mdx progenitors or by fusion of corrected satellite cells with preexisting dystrophic fibers. Immunostaining of the three types of myofibers in mdx-C mice is shown in fig. S8C.