Generation of gene-target dogs using CRISPR/Cas9 system

Zou, Qingjian; Wang, Xiaomin; Liu, Yunzhong; Ouyang, Zhen; Hai-bin, Long; Wei, Shu; Xin, Jige; Zhao, Bingzi; Lai, Sisi; Shen, Jun; Ni, Qingchun; Yang, Huaqiang; Zhong, Huilin; Li, Li; Hu, Minhua; Zhang, Quanjun; Zhou, Zhidong; He, Jiaxin; Yan, Qingyun; Fan, Nana; Zhao, Yu; Liu, Zhaoming; Guo, Lin; Huang, Jiao; Zhang, Guanguan; Jing, Ying; Lai, Liangxue; Gao, Xiang

doi:10.1093/jmcb/mjv061

Cited by 113 publications

(65 citation statements)

References 10 publications

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“…While mouse models have been widely used, the CRISPR/Cas9 gene editing approach has been established in many other animal models including worm [84], fly [85], fish [86, 87], rat [88], rabbit [89, 90], goat [91], sheep [92], dog [93], pig [94], and monkeys [95]. The expansion of transgenic animal models beyond mouse is advantageous to biomedical research that accelerates the development of new therapeutic strategies.…”

Section: Applications Of Crispr/cas9 For Cell Biological Studiesmentioning

confidence: 99%

Applications of CRISPR Genome Engineering in Cell Biology

Wang

2016

Trends in Cell Biology

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Recent advances in genome engineering are starting a revolution in biological research and translational applications. The CRISPR-associated RNA-guided endonuclease Cas9 and its variants enable diverse manipulations of genome function. In this review, we describe the development of Cas9 tools for a variety of applications in cell biology research, including the study of functional genomics, the creation of transgenic animal models, and genomic imaging. Novel genome engineering methods offer a new avenue to understand the causality between genome and phenotype, thus promising a fuller understanding of cell biology.

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Section: Applications Of Crispr/cas9 For Cell Biological Studiesmentioning

confidence: 99%

Applications of CRISPR Genome Engineering in Cell Biology

Wang

2016

Trends in Cell Biology

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“…Meanwhile, genetically modified pigs have great promise in biomedical research [14,15]. The CRISPR/Cas9 system has been demonstrated to be a highly efficient genomic targeting tool for generating gene-modified animal models [12,16,17,18,19,20]. Our previous success in generating a genetically modified pig using the CRISPR/Cas9 system inspired us to generate a DMD pig model [17,21].…”

Section: Introductionmentioning

confidence: 99%

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

Zhao

Qing

et al. 2016

IJMS

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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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“…Gene-modified sheep were produced using lentiviral vectors5 and RNA interference (RNAi)6, or through reprogrammable ZFNs7 and TALENs8, demonstrate the potential of targeting specific genes in sheep. Recently, the clustered regulatory interspaced short palindromic repeat (CRISPR)-associated (Cas)-based RNA guided DNA endonuclease such as the Streptococcus pyogenes Cas9 nuclease (CRISPR/Cas9) has enabled rapid genome editing by deleting, adding, activating, or suppressing targeted genes at a very high efficiency and specificity in a wide spectrum of organisms including human cells910, crops1112 and large animals (such as in pigs1314, goats1516, and dogs17). Cas9-mediated knockout in sheep have been demonstrated1819, opening an avenue for improving sheep breeding by genetic engineering.…”

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confidence: 99%

Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep

Wang

Niu

Zhou

et al. 2016

Sci Rep

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The CRISPR/Cas9 system provides a flexible approach for genome engineering of genetic loci. Here, we successfully achieved precise gene targeting in sheep by co-injecting one-cell-stage embryos with Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2). We carefully examined the sgRNAs:Cas9-mediated targeting effects in injected embryos, somatic tissues, as well as gonads via cloning and sequencing. The targeting efficiencies in these three genes were within the range of 27–33% in generated lambs, and that of simultaneously targeting the three genes was 5.6%, which demonstrated that micro-injection of zygotes is an efficient approach for generating gene-modified sheep. Interestingly, we observed that disruption of the MSTN gene resulted in the desired muscle hypertrophy that is characterized by enlarged myofibers, thereby providing the first detailed evidence supporting that gene modifications had occurred at both the genetic and morphological levels. In addition, prescreening for the off-target effect of sgRNAs was performed on fibroblasts before microinjection, to ensure that no detectable off-target mutations from founder animals existed. Our findings suggested that the CRISPR/Cas9 method can be exploited as a powerful tool for livestock improvement by simultaneously targeting multiple genes that are responsible for economically significant traits.

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Generation of gene-target dogs using CRISPR/Cas9 system

Cited by 113 publications

References 10 publications

Applications of CRISPR Genome Engineering in Cell Biology

Applications of CRISPR Genome Engineering in Cell Biology

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

Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep

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