In the present study, we investigated whether electroporation could be used for one-step multiplex CRISPR/Cas9-based genome editing, targeting IL2RG and GHR in 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 targeting IL2RG or one of two gRNAs targeting GHR. No significant differences in embryo development rates were found between control embryos and those subjected to electroporation, irrespective of the target gene. Two gRNAs targeting IL2RG (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 targeting GHR. In our next experiment, the mutation efficiency and the development of embryos simultaneously electroporated with gRNAs targeting IL2RG and GHR were investigated. Similar embryo development rates were observed between embryos electroporated with two gRNAs and control embryos. When IL2RG-targeting gRNA no. 2 was used with GHR-targeting gRNAs no. 1 or no. 2, a significantly higher double biallelic mutation rate was observed than with IL2RG-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.

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，对照胚胎和电穿孔胚胎之间的胚胎发育率没有显着差异。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 的胚胎的突变效率和发育。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 g​​RNA 时，没有。 2 号与GHR靶向 g​​RNA 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。