title: 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 theDMDgene 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 preciseDMDtargeting in Chinese Diannan miniature pigs by co-injecting zygotes with Cas9 mRNA and sgRNA targetingDMD. Two piglets were obtained after embryo transfer, one of piglets was identified asDMD-modified individual via traditional cloning, sequencing and T7EN1 cleavage assay. An examination of targeting rates in theDMD-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. TheDMD-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 theDMDvia CRISPR/Cas9 system in a miniature pig.
authors.affiliation: School of Life Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai 201210, China. geneyhh@126.com.|State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China. geneyhh@126.com.|College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China. geneyhh@126.com.|Research Center of Life Science, Yulin University, Yulin 719000, China. geneyhh@126.com.
authors.forename: Hong-Hao
authors.initials: HH
authors.lastname: Yu
authors.affiliation: State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China. hengzhao2014@126.com.|College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China. hengzhao2014@126.com.
authors.forename: Heng
authors.initials: H
authors.lastname: Zhao
authors.affiliation: State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China. qingyubo20@163.com.|College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China. qingyubo20@163.com.
authors.forename: Yu-Bo
authors.initials: YB
authors.lastname: Qing
authors.affiliation: College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China. pwr2000@sina.com.
authors.forename: Wei-Rong
authors.initials: WR
authors.lastname: Pan
authors.affiliation: State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China. jiabaoyu2009@163.com.|College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China. jiabaoyu2009@163.com.
authors.forename: Bao-Yu
authors.initials: BY
authors.lastname: Jia
authors.affiliation: State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China. hyzhao2000@126.com.
authors.forename: Hong-Ye
authors.initials: HY
authors.lastname: Zhao
authors.affiliation: School of Life Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai 201210, China. huangxx@shanghaitech.edu.cn.
authors.forename: Xing-Xu
authors.initials: XX
authors.lastname: Huang
authors.affiliation: State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China. hongjiangwei@126.com.|College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China. hongjiangwei@126.com.
authors.forename: Hong-Jiang
authors.initials: HJ
authors.lastname: Wei
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004195:Disease Models, Animal; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism; D004624:Embryo Transfer; D005838:Genotype; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism; D020388:Muscular Dystrophy, Duchenne / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D010641:Phenotype; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013552:Swine; D013556:Swine, Miniature; D015053:Zygote / Q000378:metabolism*
chemical_list: D016189:Dystrophin; D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger
keywords: CRISPR/Cas9; DMD; disease model; gene editing; pig