Abstract
The application of CRISPR/Cas9 strategy promises to rapidly increase the production of genetically engineered animals since it yields stably integrated transgenes. In the present study, we investigated the efficiency of target mutations after electroporation with the CRISPR/Cas9 system using sgRNAs to target the MSTN or FGF10 genes in porcine-matured oocytes and putative zygotes. Effects of pulse number (3–7 pulse repetitions) during electroporation on the embryonic development and mutation efficiency were also investigated. Our results showed that the cleavage rate of matured oocytes with electroporation treatment significantly decreased as compared with electroporated putative zygotes (p < 0.05). Moreover, the rates of blastocyst formation from oocytes/zygotes electroporated with more than 5 pulses decreased. Mutation efficiency was then assessed after sequencing the target sites in individual blastocysts derived from oocytes/zygotes electroporated by 3 and 5 pulses. No bi-allelic mutations in all examined blastocysts were observed in this study. There were no differences in the mutation rates (50–60%) between blastocysts derived from matured oocytes electroporated by 3 and 5 pulses, irrespective of targeting gene. In the targeting MSTN gene, however, the mutation rate (12.5%) of blastocysts derived from putative zygotes electroporated by 3 pulses tended to be lower than that (60%) from 5-pulsed electroporated putative zygotes. These data indicate that the type of eggs may influence not only their development after electroporation treatment but also the mutation rate in the resulting blastocysts.
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References
Brinkman EK, Chen T, Amendola M, van Steensel B (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42:e168
Foulkes W, Real F (2013) Many mosaic mutations. Curr Oncol 20:85–87
Gioia L, Barboni B, Turriani M, Capacchietti G, Pistilli MG, Berardinelli P, Mattioli M (2005) The capability of reprogramming the male chromatin after fertilization is dependent on the quality of oocyte maturation. Reproduction 130:29–39
Hashimoto M, Yamashita Y, Takemoto T (2016) Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev Biol 418:1–9
Kaneko T, Sakuma T, Yamamoto T, Mashimo T (2014) Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Sci Rep 4:6382
Legge M (1995) Oocyte and zygote zona pellucida permeability to macromolecules. J Exp Zool 271:145–150
Luo Y, Lin L, Bolund L, Jensen TG, Sorensen CB (2012) Genetically modified pigs for biomedical research. J Inherit Metab Dis 35:695–713
Nishio K, Tanihara F, Nguyen TV, Kunihara T, Nii M, Hirata M, Takemoto T, Otoi T (2018) Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos. Reprod Domest Anim 53:313–318
Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156:836–843
Onuma A, Fujii W, Sugiura K, Naito K (2017) Efficient mutagenesis by CRISPR/Cas system during meiotic maturation of porcine oocytes. J Reprod Dev 63:45–50
Semenov I, Xiao S, Pakhomov AG (2016) Electroporation by subnanosecond pulses. Biochem biophys Rep 6:253–259
Tanihara F, Takemoto T, Kitagawa E, Rao S, Do LT, Onishi A, Yamashita Y, Kosugi C, Suzuki H, Sembon S, Suzuki S, Nakai M, Hashimoto M, Yasue A, Matsuhisa M, Noji S, Fujimura T, Fuchimoto D, Otoi T (2016) Somatic cell reprogramming-free generation of genetically modified pigs. Sci Adv 2:e1600803
Umeyama K, Saito H, Kurome M, Matsunari H, Watanabe M, Nakauchi H, Nagashima H (2012) Characterization of the ICSI-mediated gene transfer method in the production of transgenic pigs. Mol Reprod Dev 79:218–228
Vilarino M, Rashid ST, Suchy FP, McNabb BR, Meulen T, Fine EJ, Ahsan S, Mursaliyev N, Sebastiano V, Diab SS (2017) CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep. Sci Rep 7:17472
Wang Y, Du Y, Shen B, Zhou X, Li J, Liu Y, Wang J, Zhou J, Hu B, Kang N (2015) Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA. Sci Rep 5:8256
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The authors would like to thank Nippon Food Packer, K. K. Shikoku (Tokushima, Japan) for supplying pig ovaries.
Funding
This work was supported in part by the Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), and by the Ministry of Education, Culture, Sports, Science, and Technology (No. 17H03938 and 17K19325).
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Editor: Tetsuji Okamoto
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Hirata, M., Tanihara, F., Wittayarat, M. et al. Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes. In Vitro Cell.Dev.Biol.-Animal 55, 237–242 (2019). https://doi.org/10.1007/s11626-019-00338-3
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DOI: https://doi.org/10.1007/s11626-019-00338-3