Quynh Anh Le1, Fuminori Tanihara2, Manita Wittayarat3, Zhao Namula1,4, Yoko Sato5, Qingyi Lin1, Koki Takebayashi1, Maki Hirata1, Takeshige Otoi1. 1. Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, 2272-1 Ishii, Myozai-gun, Tokushima, 779-3233, Japan. 2. Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, 2272-1 Ishii, Myozai-gun, Tokushima, 779-3233, Japan. tanihara@tokushima-u.ac.jp. 3. Faculty of Veterinary Science, Prince of Songkla University, Songkhla, Thailand. 4. College of Coastal Agricultural Sciences, Guangdong Ocean University, Guangdong, China. 5. School of Biological Science, Tokai University, Sapporo, Japan.
Abstract
OBJECTIVE: Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are used for generating genetically modified pigs. However, these methods create mosaic mutations in embryos. In this study, we evaluated whether the gene editing method and embryonic stage for gene editing affect the gene editing efficiency of porcine embryos. RESULTS: First, we designed five guide RNAs (gRNAs) targeting the B4GALNT2 gene and evaluated mutation efficiency by introducing each gRNA with Cas9 protein into zygotes by electroporation. Next, the optimized gRNA with Cas9 protein was introduced into 1-cell and 2-cell stage embryos by either microinjection or electroporation. The sequence of gRNA affected the bi-allelic mutation rate and mutation efficiency of blastocysts derived from electroporated embryos. Microinjection significantly decreased the cleavage rates in each embryonic stage and blastocyst formation rates in 2-cell stage embryos compared with electroporation (p < 0.05). However, the bi-allelic mutation rate and mutation efficiency of blastocysts from the 1-cell stage embryos edited using microinjection were significantly higher (p < 0.05) than those of blastocysts from the 2-cell stage embryos edited by both methods. These results indicate that the gene editing method and embryonic stage for gene editing may affect the genotype and mutation efficiency of the resulting embryos.
OBJECTIVE: Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are used for generating genetically modified pigs. However, these methods create mosaic mutations in embryos. In this study, we evaluated whether the gene editing method and embryonic stage for gene editing affect the gene editing efficiency of porcine embryos. RESULTS: First, we designed five guide RNAs (gRNAs) targeting the B4GALNT2 gene and evaluated mutation efficiency by introducing each gRNA with Cas9 protein into zygotes by electroporation. Next, the optimized gRNA with Cas9 protein was introduced into 1-cell and 2-cell stage embryos by either microinjection or electroporation. The sequence of gRNA affected the bi-allelic mutation rate and mutation efficiency of blastocysts derived from electroporated embryos. Microinjection significantly decreased the cleavage rates in each embryonic stage and blastocyst formation rates in 2-cell stage embryos compared with electroporation (p < 0.05). However, the bi-allelic mutation rate and mutation efficiency of blastocysts from the 1-cell stage embryos edited using microinjection were significantly higher (p < 0.05) than those of blastocysts from the 2-cell stage embryos edited by both methods. These results indicate that the gene editing method and embryonic stage for gene editing may affect the genotype and mutation efficiency of the resulting embryos.
Authors: T-V Nguyen; F Tanihara; Ltk Do; Y Sato; M Taniguchi; M Takagi; T Van Nguyen; T Otoi Journal: Reprod Domest Anim Date: 2017-06-28 Impact factor: 2.005
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