One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as <i>GGTA1</i>, <i>CMAH</i>, and <i>B4GALNT2</i>, are key targets to improve the outcomes of xenotransplantation...

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Bibliographic Details
Main Authors:	Fuminori Tanihara, Maki Hirata, Nhien Thi Nguyen, Osamu Sawamoto, Takeshi Kikuchi, Takeshige Otoi
Format:	Article
Language:	English
Published:	MDPI AG 2021-02-01
Series:	International Journal of Molecular Sciences
Subjects:	CRISPR/Cas9 <i>GGTA1</i> <i>CMAH</i> <i>B4GALNT2</i> pig in vitro fertilization
Online Access:	https://www.mdpi.com/1422-0067/22/5/2249

Description
Summary:	Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as <i>GGTA1</i>, <i>CMAH</i>, and <i>B4GALNT2</i>, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting <i>GGTA1</i>, <i>CMAH,</i> and <i>B4GALNT2</i> into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting <i>GGTA1</i> and <i>CMAH</i> with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when <i>GGTA1</i>, <i>CMAH</i>, and <i>B4GALNT2</i> were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of <i>GGTA1</i>/<i>CMAH</i> double-edited pigs and <i>GGTA1</i>/<i>CMAH/B4GALNT2</i> triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.
ISSN:	1661-6596 1422-0067