One-step genome editing of porcine zygotes through the electroporation of a CRISPR/Cas9 system with two guide RNAs
One-step genome editing of porcine zygotes through the electroporation of a CRISPR/Cas9 system with two guide RNAs
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DOI：
10.1007/s11626-020-00507-9
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发表时间：
2020-09-25
影响因子：
2.1
通讯作者：
Otoi, Takeshige
中科院分区：
文献类型：
--
作者：
Hirata, Maki;Wittayarat, Manita;Otoi, Takeshige
在本研究中，我们研究了电穿孔是否可用于基于 CRISPR/Cas9 的一步多重基因组编辑，针对猪胚胎中的 IL2RG 和 GHR。首先，我们通过分析囊胚形成率和基因组编辑效率来评估和选择引导RNA (gRNA)。这是在用三种不同的靶向IL2RG的gRNA之一或两种靶向GHR的gRNA之一电穿孔的胚胎中进行的。无论靶基因如何，对照胚胎和接受电穿孔的胚胎之间没有发现胚胎发育率存在显着差异。与第 1 号 gRNA 相比，两个靶向 IL2RG 的 gRNA（第 2 号和第 3 号）导致猪囊胚中双等位基因突变率增加。 1. 两种靶向GHR的gRNA之间的突变率没有显着差异。在我们的下一个实验中，研究了同时电穿孔靶向 IL2RG 和 GHR 的 gRNA 的突变效率和胚胎发育。在用两种 gRNA 电穿孔的胚胎和对照胚胎之间观察到相似的胚胎发育速率。当 IL2RG 靶向 gRNA 编号为 1 时2 号与 GHR 靶向 gRNA 一起使用。 1 或没有。如图2所示，观察到比IL2RG靶向gRNA no.2显着更高的双双等位基因突变率。 3. 总之，我们证明了使用电穿孔将多个 gRNA 和 Cas9 转移到猪受精卵中的可行性，从而实现多个基因的双双等位基因突变，从而具有良好的胚胎存活率。
In the present study, we investigated whether electroporation could be used for one-step multiplex CRISPR/Cas9-based genome editing, targetingIL2RGandGHRin porcine embryos. First, we evaluated and selected guide RNAs (gRNAs) by analyzing blastocyst formation rates and genome editing efficiency. This was performed in embryos electroporated with one of three different gRNAs targetingIL2RGor one of two gRNAs targetingGHR. No significant differences in embryo development rates were found between control embryos and those subjected to electroporation, irrespective of the target gene. Two gRNAs targetingIL2RG(nos. 2 and 3) contributed to an increased biallelic mutation rate in porcine blastocysts compared with gRNA no. 1. There were no significant differences in the mutation rates between the two gRNAs targetingGHR. In our next experiment, the mutation efficiency and the development of embryos simultaneously electroporated with gRNAs targetingIL2RGandGHRwere investigated. Similar embryo development rates were observed between embryos electroporated with two gRNAs and control embryos. WhenIL2RG-targeting gRNA no. 2 was used withGHR-targeting gRNAs no. 1 or no. 2, a significantly higher double biallelic mutation rate was observed than withIL2RG-targeting gRNA no. 3. In conclusion, we demonstrate the feasibility of using electroporation to transfer multiple gRNAs and Cas9 into porcine zygotes, enabling the double biallelic mutation of multiple genes with favorable embryo survival.