Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes

Abstract

CD163 is a putative fusion receptor for virus of porcine reproductive and respiratory syndrome (PRRS). In this study, we introduced a CRISPR/Cas9 system [guide RNAs (gRNAs) with Cas9 protein] targeting the CD163 gene into in vitro-fertilized porcine zygotes by electroporation to generate CD163-modified pigs. First, we designed four types of gRNAs that targeted distinct sites in exon 7 of the CD163 gene. Cas9 protein with different gRNAs was introduced into in vitro-fertilized zygotes by electroporation. When the electroporated zygotes were allowed to develop to blastocysts in vitro and the genome editing efficiency was evaluated using these blastocysts, three (gRNA1, 2, and 4) of the four gRNAs tested successfully edited the CD163 gene. To generate CD163-knockout pigs, a total of 200 electroporated zygotes using these three gRNAs were transferred into the oviducts of oestrous-synchronized surrogate and the surrogate gave birth to eight piglets. Subsequent sequence analysis revealed that one of the piglets carried no wild-type sequence in CD163 gene. The other seven piglets carried only wild-type sequence. Thus, we successfully generated a CD163-edited pig by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes, although further improvement is required to generate genetically modified pigs with high efficiency.

Keywords:

• CD163
• CRISPR/Cas9
• electroporation; in vitro fertilization
• porcine reproductive and respiratory syndrome

Acknowledgments

The authors would like to thank Dr. Taro Mito (Tokushima University) for analysis of porcine genotype, and Nippon Food Packer and K. K. Shikoku (Tokushima, Japan) for supplying pig ovaries.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was supported in part by the ‘Funds for the Development of Human Resources in Science and Technology’ under MEXT, through the ‘Home for Innovative Researchers and Academic Knowledge Users (HIRAKU)’ Consortium and JSPS KAKENHI [grant nos. JP17H03938, JP18K12062 and JP19K16014]. We acknowledge Tokushima University for their financial support of the Research Clusters program of Tokushima University [grant No. 1701001].