Genome edited animals can now be easily produced using the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system. Traditionally, these animals have been produced by the introduction of endonucleases into pronuclear-stage embryos. Recently, a novel electroporation method, the "Technique for Animal Knockout system by Electroporation (TAKE)," has been established as a simple and highly efficient tool to introduce endonucleases into embryos instead of methods such as microinjection. Use of frozen-warmed pronuclear-stage embryos in this method has further contributed to efficient production of genome edited animals. However, early developmental stage embryos, including pronuclear-stage embryos, especially those of rats, sometimes show low resistance to physical damage by vitrification and introduction of endonucleases during microinjection. In this study, we propose an ethanol-free, slow-freezing method to reduce physical damage to pronuclear-stage embryos followed by the TAKE method. All mouse and rat frozen embryos were survived after electroporation, and 18% and 100% of offspring were edited target gene, respectively. The resulting protocol is an efficient method for producing genome edited animals.

中文翻译：

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通过将 CRISPR/Cas9 电穿孔到冷冻温热的原核阶段胚胎对啮齿动物进行基因组编辑

现在可以使用成簇的规则间隔短回文重复序列 (CRISPR) 和 CRISPR 相关蛋白 9 (Cas9) 系统轻松生成基因组编辑的动物。传统上，这些动物是通过将核酸内切酶引入原核阶段胚胎来生产的。最近，一种新的电穿孔方法，即“通过电穿孔 (TAKE) 进行动物敲除系统的技术”，已被确立为一种简单且高效的工具，可将核酸内切酶引入胚胎中，而不是显微注射等方法。在这种方法中使用冷冻加热的原核阶段胚胎进一步有助于基因组编辑动物的有效生产。然而，早期发育阶段的胚胎，包括原核阶段的胚胎，尤其是大鼠的胚胎，有时在显微注射过程中对玻璃化和内切核酸酶的引入对物理损伤的抵抗力较低。在这项研究中，我们提出了一种无乙醇的慢速冷冻方法，以减少对原核阶段胚胎的物理损伤，然后是 TAKE 方法。电穿孔后所有小鼠和大鼠冷冻胚胎均存活，后代分别有18%和100%的靶基因被编辑。由此产生的协议是一种生产基因组编辑动物的有效方法。