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.
Highlights
The DMD mutation is the genetic factor responsible for the development of Duchenne muscular dystrophy (DMD), an X-linked hereditary muscle dystrophy [1,2]
The Diannan miniature pig is well known as an exclusive native breed in Yunnan Province, China
These pigs are famous for their early sexual maturation and suitable full-grown body weight, which makes this strain ideal for generating a human disease model [22,23]
Summary
The DMD mutation is the genetic factor responsible for the development of Duchenne muscular dystrophy (DMD), an X-linked hereditary muscle dystrophy [1,2]. Several animal models manifesting the phenotype observed in DMD disease have been generated in the laboratory or identified in nature, including mdx mice and dogs with X-linked muscular dystrophy (cxmd) [7]. These models generally show the pathological alterations observed in human patients and have been used to understand the pathological mechanism of DMD and to test candidate therapies [8,9]. Pigs are an ideal animal model for human disease because their physiological and anatomical characteristics are more similar to humans than are those of mice, rats and dogs These similarities increase the likelihood of a more accurate recapitulation of the DMD. We employed the CRISPR/Cas system to knockout DMD to determine whether pigs lacking dystrophin could function as an animal model by recapitulating the human DMD phenotypes
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