Generation of <i>CD163-</i>edited pig via electroporation of the CRISPR/Cas9 system into porcine <i>in vitro-</i>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 <i>CD163</i> gene into <i>in vitro</i>-fertilized porcine zygotes by electroporation to generate <i>CD163</i>-modified pigs. First, we designed four types of gRNAs that targeted distinct sites in exon 7 of the <i>CD163</i> gene. Cas9 protein with different gRNAs was introduced into <i>in vitro</i>-fertilized zygotes by electroporation. When the electroporated zygotes were allowed to develop to blastocysts <i>in vitro</i> and the genome editing efficiency was evaluated using these blastocysts, three (gRNA1, 2, and 4) of the four gRNAs tested successfully edited the <i>CD163</i> gene. To generate <i>CD163</i>-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 <i>CD163</i> gene. The other seven piglets carried only wild-type sequence. Thus, we successfully generated a <i>CD163</i>-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.