Fuminori Tanihara 1 , 2 , Tatsuya Takemoto 3 , † , Eri Kitagawa 4 , Shengbin Rao 4 , Lanh Thi Kim Do 1 , Akira Onishi 5 , 6 , Yukiko Yamashita 3 , Chisato Kosugi 3 , Hitomi Suzuki 3 , Shoichiro Sembon 6 , Shunichi Suzuki 6 , Michiko Nakai 6 , Masakazu Hashimoto 7 , Akihiro Yasue 8 , Munehide Matsuhisa 2 , Sumihare Noji 9 , Tatsuya Fujimura 4 , Dai-ichiro Fuchimoto 6 , Takeshige Otoi 1 , †
14 September 2016
A new and highly efficient method for generating mutant pigs by electroporating the CRISPR/Cas9 system into zygotes.
Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.