Genome-Wide PERV Inactivation in Pigs Using CRISPR/Cas9

The shortage of organs for transplantation is probably the biggest unmet medical need. A potential problem with the clinical use of porcine xenografts is the risk that porcine endogenous retroviruses (PERVs) could infect human cells. In the past, we determined the PERV copy number in the porcine kidney epithelial cell line PK15 and in primary fibroblasts. Using CRISPR-Cas9, we disrupted the catalytic center of pol, which is essential for virus replication. Next, we isolated cells in which 100% of the PERV elements had been inactivated. This method enables the possibility of eradicating PERVs in vitro for application to pig-to-human xenotransplantation. Here we describe the methodological bases of this work.