論文

国際誌
2020年7月

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

Molecular biology reports
  • Maki Hirata
  • ,
  • Manita Wittayarat
  • ,
  • Zhao Namula
  • ,
  • Quynh Anh Le
  • ,
  • Qingyi Lin
  • ,
  • Nhien Thi Nguyen
  • ,
  • Koki Takebayashi
  • ,
  • Yoko Sato
  • ,
  • Fuminori Tanihara
  • ,
  • Takeshige Otoi

47
7
開始ページ
5073
終了ページ
5079
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1007/s11033-020-05576-3

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.

リンク情報
DOI
https://doi.org/10.1007/s11033-020-05576-3
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/32519310
ID情報
  • DOI : 10.1007/s11033-020-05576-3
  • PubMed ID : 32519310

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