Triple gene editing in porcine embryos using electroporation alone or in combination with microinjection

• Published in: Veterinary World 2022-02, Vol.15 (2), p.496-501
• Main Authors: Namula, Zhao, Le, Quynh Anh, Wittayarat, Manita, Lin, Qingyi, Takebayashi, Koki, Hirata, Maki, Do, Lanh Thi Kim, Tanihara, Fuminori, Otoi, Takeshige
• Format: Article
• Language: English
• Subjects: clustered regularly interspaced short palindromic repeats/cas9; electroporation; gene editing; microinjection; porcine zygotes
• ISSN: 0972-8988; 2231-0916
• DOI: 10.14202/vetworld.2022.496-501

We previously developed the gene-editing by electroporation (EP) of Cas9 protein method, in which the CRISPR/Cas9 system was introduced into porcine fertilized (IVF) zygotes through EP to disrupt a target gene. This method should be further developed, and a combination of EP and MI methods should be evaluated in pigs. This study aimed to determine that a combination of microinjection (MI) and EP of CRISPR/Cas9 system could increase the rates of biallelic mutation for triple-gene knockout in porcine blastocysts. We targeted the pancreatic and duodenal homeobox1 ( ) gene using cytoplasmic MI 1 h before or after EP, which was used to edit alpha-1,3-galactosyltransferase ( ) and cytidine 32 monophosphate-N-acetylneuraminic acid hydroxylase ( ) genes in porcine zygotes. We introduced guide RNAs targeting , , and with the Cas9 protein into IVF zygotes (one-cell stage) through EP 10 h after the start of IVF (IVF; EP group) or in combination with MI (1 h before, MI-EP group, or after EP treatment EP-MI group) and evaluated the blastocyst formation rate and efficiency of target mutations in the resulting blastocysts. Our results revealed a significant reduction in the rate of blastocyst formation in the two groups that underwent MI before and after EP (MI-EP and EP-MI group), compared with that in the groups treated with EP alone (EP group) (p=0.0224 and p