論文

2019年6月14日

Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos.

The Journal of reproduction and development
  • Fuminori Tanihara
  • ,
  • Maki Hirata
  • ,
  • Nhien Thi Nguyen
  • ,
  • Quynh Anh LE
  • ,
  • Takayuki Hirano
  • ,
  • Takeshige Otoi

65
3
開始ページ
209
終了ページ
214
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1262/jrd.2018-116

Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/Cas9 components for introduction into oocytes/zygotes by CI, to reduce mosaicism in the resulting blastocysts. First, we introduced 20 ng/μl of Cas9 protein and guide RNA (gRNA), targeting the α-1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes/zygotes before and after IVF, twice. CI treatment had no detrimental effects on blastocyst formation rates. The highest value of the rate of mutant blastocysts was observed in zygotes injected after IVF. Next, we injected Cas9 protein and gRNA into zygotes after IVF at a concentration of 20 ng/μl each (20 ng/μl group) or 100 ng/μl each (100 ng/μl group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/μl group was significantly higher (P < 0.05) than that in the 20 ng/μl group. Although no blastocysts from the 20 ng/μl group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/μl group carried a biallelic mutation. In conclusion, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI, and the concentration of CRISPR/Cas9 components, is needed.

リンク情報
DOI
https://doi.org/10.1262/jrd.2018-116
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/30726783
PubMed Central
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584178
ID情報
  • DOI : 10.1262/jrd.2018-116
  • PubMed ID : 30726783
  • PubMed Central 記事ID : PMC6584178

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