Gene editing in porcine embryos using a combination of electroporation and transfection methods
Gene editing in porcine embryos using a combination of electroporation and transfection methods
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结合电穿孔和转染方法对猪胚胎进行基因编辑
DOI：
10.1111/rda.14184
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发表时间：
2022
影响因子：
1.7
通讯作者：
Otoi Takeshige
中科院分区：
文献类型：
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作者：
Takebayashi Koki;Wittayarat Manita;Lin Qingyi;Hirata Maki;Yoshimura Naoaki;Torigoe Nanaka;Nagahara Megumi;Do Lanh Thi Kim;Tanihara Fuminori;Otoi Takeshige
CRISPR/Cas9(簇状规则间隔短回文重复序列-CRISPR相关蛋白9)技术正在迅速发展，并在很大程度上影响了不同应用中遗传修改的效率和效果。在CRISPR/CAS9系统的开发中，一个越来越重要的研究方面是将CRISPR材料引入目标生物。尽管我们先前证明了电穿孔和脂质体介导的CRISPR/Cas9基因破坏在猪受精卵中的有效性，但我们仍然相信将这两种方法结合起来可以提高该系统的效率。因此，本研究旨在阐明电穿孔和脂质体联合作用将CRISPR/Cas9组分转移到透明带(ZP)完整和无受精卵中的效果。结果显示，与单独脂质体相比，单独电穿孔显著增加了所产生的囊胚中的双等位基因突变率，而不考虑ZP的存在。单独用脂质体处理的ZP完整受精卵没有任何突变，这表明去除ZP对于通过脂质体处理在受精卵中进行基于CRISPR/Cas9的基因组编辑是必要的。电穿孔后的额外脂质体处理并没有提高ZP完整或不含ZP的受精卵所产生的囊胚的总突变率和双等位基因突变率。
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats‐CRISPR‐associated protein 9) technology is growing rapidly and has been greatly influencing the efficiency and effectiveness of genetic modifications in different applications. One aspect of research gaining importance in the development of the CRISPR/Cas9 system is the introduction of CRISPR materials into target organisms. Although we previously demonstrated the efficacy of electroporation‐ and lipofection‐mediated CRISPR/Cas9 gene disruption in porcine zygotes, we still believe that the efficiency of this system could be improved by combining these two methods. The present study was thus conducted to clarify the effects of a combination of electroporation and lipofection for delivering CRISPR/Cas9 components into zona pellucida (ZP)‐intact and ‐free zygotes. The results revealed that electroporation alone significantly increased the biallelic mutation rates in the resulting blastocysts compared to lipofection alone, irrespective of the presence of ZP. None of ZP‐intact zygotes treated by lipofectamine alone had any mutations, suggesting that removal of the ZP is necessary for enabling CRISPR/Cas9‐based genome editing via lipofection treatment in the zygotes. Additional lipofectamine treatment after electroporation did not improve the rates of total and biallelic mutations in the resulting blastocysts derived from either ZP‐intact or ‐free zygotes.