Tanihara Fuminori, Hirata Maki, Nguyen Nhien T, Le Quynh A, Hirano Takayuki, Takemoto Tatsuya, Nakai Michiko, Fuchimoto Dai-Ichiro, Otoi Takeshige
Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, Ishii-cho, Tokushima, Japan.
Division of Embryology, Institute of Advanced Medical Sciences, Tokushima University, Kuramoto-cho, Tokushima, Japan.
Anim Sci J. 2019 Jan;90(1):55-61. doi: 10.1111/asj.13129. Epub 2018 Oct 25.
Recently, we established the GEEP ("gene editing by electroporation of Cas9 protein") method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome-editing efficiency. To produce an animal model for diabetes, we targeted PDX-1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX-1 exon 1 was introduced into in vitro-fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX-1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX-1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.
最近，我们建立了GEEP（“通过电穿孔导入Cas9蛋白进行基因编辑”）方法，该方法是通过电穿孔将由Cas9蛋白和单向导RNA（sgRNA）组成的CRISPR/Cas9系统导入猪受精卵中，从而诱导高效的靶向基因破坏。在本研究中，我们检测了sgRNA对猪胚胎囊胚形成的影响，并评估了它们的基因组编辑效率。为了构建糖尿病动物模型，我们靶向了PDX-1（胰腺十二指肠同源盒1）基因，该基因在胎儿期胰腺发育中至关重要，其单等位基因破坏会损害胰岛素分泌。首先，通过GEEP方法将携带靶向PDX-1外显子1不同位点的不同sgRNA的Cas9蛋白导入体外受精的受精卵中。在测试的六种sgRNA中，三种sgRNA（sgRNA1、2和3）成功修饰了PDX-1基因。用sgRNA3编辑的受精卵的囊胚形成率显著低于（p < 0.05）未进行电穿孔处理的对照受精卵。我们的研究表明，GEEP方法可成功用于生成PDX-1突变囊胚，但受精卵基因组编辑的发育情况和效率可能会受到用于CRISPR/Cas9系统的sgRNA的影响。
