Generation of Edited Pig Via Electroporation of the CRISPR/Cas9 System into Porcine Fertilized Zygotes

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