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

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 do...

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Autores principales: Yu, Hong-Hao, Zhao, Heng, Qing, Yu-Bo, Pan, Wei-Rong, Jia, Bao-Yu, Zhao, Hong-Ye, Huang, Xing-Xu, Wei, Hong-Jiang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085701/
https://www.ncbi.nlm.nih.gov/pubmed/27735844
http://dx.doi.org/10.3390/ijms17101668
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author Yu, Hong-Hao
Zhao, Heng
Qing, Yu-Bo
Pan, Wei-Rong
Jia, Bao-Yu
Zhao, Hong-Ye
Huang, Xing-Xu
Wei, Hong-Jiang
author_facet Yu, Hong-Hao
Zhao, Heng
Qing, Yu-Bo
Pan, Wei-Rong
Jia, Bao-Yu
Zhao, Hong-Ye
Huang, Xing-Xu
Wei, Hong-Jiang
author_sort Yu, Hong-Hao
collection PubMed
description 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.
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spelling pubmed-50857012016-11-01 Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy Yu, Hong-Hao Zhao, Heng Qing, Yu-Bo Pan, Wei-Rong Jia, Bao-Yu Zhao, Hong-Ye Huang, Xing-Xu Wei, Hong-Jiang Int J Mol Sci Article 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. MDPI 2016-10-09 /pmc/articles/PMC5085701/ /pubmed/27735844 http://dx.doi.org/10.3390/ijms17101668 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Yu, Hong-Hao
Zhao, Heng
Qing, Yu-Bo
Pan, Wei-Rong
Jia, Bao-Yu
Zhao, Hong-Ye
Huang, Xing-Xu
Wei, Hong-Jiang
Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy
title Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy
title_full Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy
title_fullStr Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy
title_full_unstemmed Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy
title_short Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy
title_sort porcine zygote injection with cas9/sgrna results in dmd-modified pig with muscle dystrophy
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085701/
https://www.ncbi.nlm.nih.gov/pubmed/27735844
http://dx.doi.org/10.3390/ijms17101668
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