Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation
Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation
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DOI：
10.1111/asj.13129
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发表时间：
2019-01-01
影响因子：
2
通讯作者：
Otoi, Takeshige
中科院分区：
文献类型：
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作者：
Tanihara, Fuminori;Hirata, Maki;Otoi, Takeshige
最近，我们建立了GEEP（“通过Cas9蛋白的电穿孔进行基因编辑”）方法，其中通过电穿孔将由Cas9蛋白和单向导RNA（sgRNA）组成的CRISPR/Cas9系统引入猪受精卵中，从而诱导高效的靶向基因破坏。在这项研究中，我们研究了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。为了产生糖尿病的动物模型，我们靶向PDX-1（胰腺十二指肠同源框1），这是一种在胎儿时期对胰腺发育至关重要的基因，其单等位基因破坏会损害胰岛素分泌。首先，通过GEEP方法将具有靶向PDX-1外显子1中的不同位点的不同sgRNA的Cas9蛋白引入体外受精的合子中。在测试的六种sgRNA中，三种sgRNA（sgRNA 1、2和3）成功修饰PDX-1基因。用sgRNA 3编辑的受精卵的囊胚形成率显著（p < 0.05）低于未经电穿孔处理的对照受精卵。我们的研究表明，GEEP方法可以成功地用于产生PDX-1突变体囊胚，但是用于CRISPR/Cas9系统的sgRNA可能会影响受精卵的发育和编辑基因组的效率。
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.