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Int. J. Mol. Sci. 2016, 17(10), 1668; doi:10.3390/ijms17101668

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

1
School of Life Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai 201210, China
2
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China
3
College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
4
Research Center of Life Science, Yulin University, Yulin 719000, China
These authors contribute equally to this work.
*
Authors to whom correspondence should be addressed.
Academic Editor: Julian Borejdo
Received: 19 July 2016 / Revised: 21 September 2016 / Accepted: 23 September 2016 / Published: 9 October 2016
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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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. View Full-Text
Keywords: CRISPR/Cas9; DMD; pig; disease model; gene editing CRISPR/Cas9; DMD; pig; disease model; gene editing
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Yu, H.-H.; Zhao, H.; Qing, Y.-B.; Pan, W.-R.; Jia, B.-Y.; Zhao, H.-Y.; Huang, X.-X.; Wei, H.-J. Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy. Int. J. Mol. Sci. 2016, 17, 1668.

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