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Evaluation of multiple gene targeting in porcine embryos by the CRISPR/Cas9 system using electroporation

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Abstract

The CRISPR/Cas9 system now allows for unprecedented possibilities of genome editing. However, there are some limitations, including achieving efficient one-step multiple genome targeting to save costs, time, and ensure high quality. In the present study, we investigated the efficiency of one-step multiple gene modification by electroporation in porcine zygotes using pooled guide RNAs (gRNAs) targeting CMAH, GHR, GGTA1, and PDX1. We first selected the best-performing gRNA from three different designs for each gene based on the effect on embryo development and mutation efficiency. The three gRNAs showed equivalent effects on the rates of blastocyst formation in each targeted gene; however, gRNAs CMAH #2, GHR #3, GGTA1 #3, and PDX1 #3 showed the highest biallelic mutation rate, although the total mutation rate of PDX1 #3 was significantly lower than that of PDX1 #1. Therefore, CMAH #2, GHR #3, GGTA1 #3, and PDX1 #1 were used as a mixture in electroporation to further clarify whether multiple genes can be targeted simultaneously. Individual sequencing of 43 blastocysts at the target sites of each gene showed mutations in one and two target genes in twenty-four (55.8%) and nine (20.9%) blastocysts, respectively. No mutation was detected in any target gene in ten (23.3%) blastocysts and no blastocysts had a mutation in three or more target genes. These results indicate that electroporation could effectively deliver multiple gRNAs and Cas9 protein into porcine zygotes to target multiple genes in a one-step process. However, the technique requires further development to increase the success rate of multiple gene modification.

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The datasets generated and analyzed during the current study are included in the manuscript.

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Acknowledgements

We thank the Nippon Food Packer, K. K. Shikoku (Tokushima, Japan) for supplying the pig ovaries. This study was supported in part by the Program of Open Innovation Platform with Enterprises, Research Institute and Academia (OPERA) Grant Number JPMJOP1613 from the Japan Science and Technology Agency (JST), and KAKENHI Grant Numbers JP17H03938, JP18K12062 and JP19K16014 from the Japan Society for the Promotion of Science (JSPS). We acknowledge Tokushima University for their financial support of the Research Clusters program of Tokushima University (No. 1701001).

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Authors and Affiliations

  1. Faculty of Bioscience and Bioindustry, Tokushima University, 2272-1 Ishii, Myozai-gun, Tokushima, 779-3233, Japan

    Maki Hirata, Quynh Anh Le, Qingyi Lin, Nhien Thi Nguyen, Koki Takebayashi, Fuminori Tanihara & Takeshige Otoi

  2. Faculty of Veterinary Science, Prince of Songkla University, Songkhla, Thailand

    Manita Wittayarat

  3. Faculty of Veterinary Science, Guangdong Ocean University, Zhanjiang, China

    Zhao Namula

  4. School of Biological Science, Tokai University, Sapporo, Japan

    Yoko Sato

Authors
  1. Maki Hirata
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  2. Manita Wittayarat
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  3. Zhao Namula
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  4. Quynh Anh Le
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  5. Qingyi Lin
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  6. Nhien Thi Nguyen
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  7. Koki Takebayashi
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  8. Yoko Sato
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  9. Fuminori Tanihara
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  10. Takeshige Otoi
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Contributions

MH, FT, and TO conceived the study and wrote the manuscript. MH, FT, and ZN performed the majority of experiments. TO designed the study, coordinated all of the experiments, and reviewed the manuscript. QAL, QL, NTN, and KT contributed to the laboratory work and statistical analysis. MW and YS revised the manuscript. All of the authors read and accepted the manuscript.

Corresponding author

Correspondence to Fuminori Tanihara.

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The authors declare that they have no conflict of interest.

Ethics approval

All procedures involving the handling and collection of materials from animals were approved by the Animal Research Committee of Tokushima University (Approval Number: T28-21), and were performed in accordance with the Guidelines for Animal Experiments of Tokushima University based on the rules of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) JAPAN, and the Ministry of the Environment.

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Hirata, M., Wittayarat, M., Namula, Z. et al. Evaluation of multiple gene targeting in porcine embryos by the CRISPR/Cas9 system using electroporation. Mol Biol Rep 47, 5073–5079 (2020). https://doi.org/10.1007/s11033-020-05576-3

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  • DOI: https://doi.org/10.1007/s11033-020-05576-3

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