Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy

Abstract

Dystrophinopathy, including Duchenne muscle dystrophy (DMD) and Becker muscle dystrophy (BMD) is an incurable X-linked hereditary muscle dystrophy caused by a mutation in the DMD gene in coding dystrophin. Advances in further understanding DMD/BMD for therapy are expected. Studies on mdx mice and dogs with muscle dystrophy provide limited insight into DMD disease mechanisms and therapeutic testing because of the different pathological manifestations. Miniature pigs share similar physiology and anatomy with humans and are thus an excellent animal model of human disease. Here, we successfully achieved precise DMD targeting in Chinese Diannan miniature pigs by co-injecting zygotes with Cas9 mRNA and sgRNA targeting DMD. Two piglets were obtained after embryo transfer, one of piglets was identified as DMD-modified individual via traditional cloning, sequencing and T7EN1 cleavage assay. An examination of targeting rates in the DMD-modified piglet revealed that sgRNA:Cas9-mediated on-target mosaic mutations were 70% and 60% of dystrophin alleles in skeletal and smooth muscle, respectively. Meanwhile, no detectable off-target mutations were found, highlighting the high specificity of genetic modification using CRISPR/Cas9. The DMD-modified piglet exhibited degenerative and disordered phenotypes in skeletal and cardiac muscle, and declining thickness of smooth muscle in the stomach and intestine. In conclusion, we successfully generated myopathy animal model by modifying the DMD via CRISPR/Cas9 system in a miniature pig.

Keywords: CRISPR/Cas9; DMD; disease model; gene editing; pig.

Figure 1

Evaluation of DMD sgRNA:Cas9-mediated modifications of DMD in pig parthenogenetic embryos. (a) Schematic diagram of the pig DMD partial protein coding region and the DMD sgRNA:Cas9 targeting site; (b) Polymerase chain reaction (PCR) products of the DMD targeting region in pig embryos. MK, DNA marker; Con, PCR product of the targeted region amplified from wild-type pig; (c) Detection of sgRNA:Cas9-mediated on-target cleavage of DMD by T7EN1 cleavage assay in pig embryos; (d) Representative sequencing chromatographs of PCR product. Blue background indicates the DMD sgRNA sequence. Red triangle indicates cleavage site mediated by DMD sgRNA:Cas9. Green background indicates PAM construct.

Figure 2

Detection of DMD sgRNA:Cas9-mediated modifications of DMD in funicle tissues of founder pigs. (a) Photograph of newborn founders; (b) DMD-modified founder A; (c) PCR products of the DMD targeted region amplified from founder pigs. MK, DNA marker; A, founder A; B, founder B; Con, PCR product of targeted region amplified from wild-type pig; (d) Detection of sgRNA:Cas9-mediated on-target cleavage of DMD by the T7EN1 cleavage assay in founder pigs. Con, PCR product of targeted region amplified from wild-type pig, digested by T7EN1.

Figure 3

sgRNA:Cas9-mediated DMD modifications in different tissues of founder A. (a) PCR products of the DMD targeting region. WT, PCR product amplified from wild-type pig; (b) Detection of sgRNA:Cas9-mediated on-target cleavage of DMD by the T7EN1 cleavage assay; (c) Modified DMD sequences detected in founder A. WT, wild-type DNA sequence. Sequences complementary to sgRNA targeting site are labeled in red, and PAM sequences are in green; the mutations in blue, lower case indicates the inserted base, deletions (−), insertions (+); (d) sgRNA:Cas9-mediated DMD modification frequency in different founder A tissues. Mutant 1, 11 base pair deletion; Mutant 2, 36 base pair deletion; Mutant 3, five base pair deletion and 14 base pair insertion; Mutant 4, 6 base pair deletion and 16 base pair insertion.

Figure 4

Expression of dystrophin in muscle tissues. (a) The relative expression of DMD mRNA in founder A and the age-matched wild-type pigs; (b) Western blotting for Dystrophin and GAPDH in biceps femoris muscle, heart and intestine of DMD-modified founder A and WT; (c) Immunostaining for dystrophin in biceps femoris muscle, heart and intestinal of DMD-modified founder A and WT. A indicates founder A, WT indicates wild-type pig of the same age. Frozen section. Scale bar: 50 µm.

Figure 4

Expression of dystrophin in muscle tissues. (a) The relative expression of DMD mRNA in founder A and the age-matched wild-type pigs; (b) Western blotting for Dystrophin and GAPDH in biceps femoris muscle, heart and intestine of DMD-modified founder A and WT; (c) Immunostaining for dystrophin in biceps femoris muscle, heart and intestinal of DMD-modified founder A and WT. A indicates founder A, WT indicates wild-type pig of the same age. Frozen section. Scale bar: 50 µm.

Figure 5

DMD deficiency resulted in pathological alterations in the DMD-modified pig. (a) The birth weight (480 g) and body weight at 52 days (7809 g) of founder A were lower than that of the wild-type piglet (birth weight 575 g and body weight at 52 days was 110,200 g); (b) Behavioral performance of muscle weakness of founder A; (c) Mean minimal Feret’s diameter of muscle fibers in biceps femoris muscle of 52-day-old founder A and WT; (d) Proportion of muscle fibers with central nuclei in deltoid muscle of 52-day-old founder A and WT. ** p < 0.01; (e–h,j–o) Haematoxylin and eosin (H&E) staining, paraffin section; (e–i) Smooth muscle thickness of stomach (e) and intestine (g) were dramatically decreased compared with that of WT (f,h). Blue arrows indicate the thickness of stomach and intestine; (j) Cross-section of biceps femoris muscle revealed disordered myofiber construction and the green arrow indicated the aggregated nuclei; (l) Black arrows indicated the rounded myofibers with central nuclei, and red arrows indicated the necrosis of myofibers in deltoid muscle; (n) Multifocal area of pale discoloration in cardiac muscle was observed in section; (k,m,o) histological section of skeletal muscle and cardiac muscle of wild-type. Scale bars: 50 µm (e–h,j–m),100 µm (n,o).