Effects of electroporation treatment using different concentrations of Cas9 protein with gRNA targeting <i>Myostatin</i> (<i>MSTN</i>) genes on the development and gene editing of porcine zygotes
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- Quynh A. Le
- Faculty of Bioscience and Bioindustry Tokushima University Tokushima Japan
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- Maki Hirata
- Faculty of Bioscience and Bioindustry Tokushima University Tokushima Japan
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- Nhien T. Nguyen
- Faculty of Bioscience and Bioindustry Tokushima University Tokushima Japan
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- Koki Takebayashi
- Faculty of Bioscience and Bioindustry Tokushima University Tokushima Japan
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- Manita Wittayarat
- Faculty of Veterinary Science Prince of Songkla University Songkhla Thailand
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- Yoko Sato
- School of Biological Science Tokai University Sapporo Japan
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- Zhao Namula
- Faculty of Veterinary Science Guangdong Ocean University Zhanjiang China
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- Masahiro Nii
- Tokushima Prefectural Livestock Research Institute Tokushima Japan
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- Fuminori Tanihara
- Faculty of Bioscience and Bioindustry Tokushima University Tokushima Japan
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- Takeshige Otoi
- Faculty of Bioscience and Bioindustry Tokushima University Tokushima Japan
説明
<jats:title>Abstract</jats:title><jats:p>This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000 ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off‐target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting <jats:italic>Myostatin</jats:italic> genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25 ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off‐target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non‐specific cleavage of off‐target sites.</jats:p>
収録刊行物
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- Animal Science Journal
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Animal Science Journal 91 (1), e13386-, 2020-01
Wiley