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Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes

  • Maki Hirata
  • Fuminori TaniharaEmail author
  • Manita Wittayarat
  • Takayuki Hirano
  • Nhien Thi Nguyen
  • Quynh Anh Le
  • Zhao Namula
  • Masahiro Nii
  • Takeshige Otoi
Article

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.

Keywords

CRISPR/Cas9 Electroporation In vitro fertilization Mosaic Pig 

Notes

Acknowledgments

The authors would like to thank Nippon Food Packer, K. K. Shikoku (Tokushima, Japan) for supplying pig ovaries.

Funding information

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).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

References

  1. Brinkman EK, Chen T, Amendola M, van Steensel B (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42:e168CrossRefGoogle Scholar
  2. Foulkes W, Real F (2013) Many mosaic mutations. Curr Oncol 20:85–87CrossRefGoogle Scholar
  3. 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 130:29–39CrossRefGoogle Scholar
  4. 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 418:1–9CrossRefGoogle Scholar
  5. Kaneko T, Sakuma T, Yamamoto T, Mashimo T (2014) Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Sci Rep 4:6382CrossRefGoogle Scholar
  6. Legge M (1995) Oocyte and zygote zona pellucida permeability to macromolecules. J Exp Zool 271:145–150CrossRefGoogle Scholar
  7. Luo Y, Lin L, Bolund L, Jensen TG, Sorensen CB (2012) Genetically modified pigs for biomedical research. J Inherit Metab Dis 35:695–713CrossRefGoogle Scholar
  8. 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 53:313–318CrossRefGoogle Scholar
  9. 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 156:836–843CrossRefGoogle Scholar
  10. Onuma A, Fujii W, Sugiura K, Naito K (2017) Efficient mutagenesis by CRISPR/Cas system during meiotic maturation of porcine oocytes. J Reprod Dev 63:45–50CrossRefGoogle Scholar
  11. Semenov I, Xiao S, Pakhomov AG (2016) Electroporation by subnanosecond pulses. Biochem biophys Rep 6:253–259Google Scholar
  12. 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:e1600803CrossRefGoogle Scholar
  13. 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 79:218–228CrossRefGoogle Scholar
  14. 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 7:17472CrossRefGoogle Scholar
  15. 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 5:8256CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  1. 1.Faculty of Bioscience and BioindustryTokushima UniversityTokushimaJapan
  2. 2.Faculty of Veterinary SciencePrince of Songkla UniversitySongkhlaThailand
  3. 3.Faculty of Veterinary ScienceGuangdong Ocean UniversityZhanjiangChina
  4. 4.Tokushima Prefectural Livestock Research InstituteTokushimaJapan

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