The Relationship between Embryonic Development and the Efficiency of Target Mutations in Porcine Endogenous Retroviruses (PERVs) Pol Genes in Porcine Embryos

Porcine endogenous retrovirus (PERV) is a provirus found in the pig genome that may act as an infectious pathogen in humans who receive pig organ xenotransplantation. Inactivation of the PERV pol gene in porcine cells reportedly affects cell growth. Therefore, the mutation of PERV pol gene in porcine embryos using genome editing may affect the embryonic development. The present study was carried out to investigate the relationship between the mutation of the PERV pol gene in porcine embryos and their development. We introduced, either alone or in combination, three different gRNAs (gRNA1, 2, and 3) into porcine zygotes by genome editing using electroporation of the Cas9 protein (GEEP) system. All three gRNAs targeted the PERV pol gene, and we assessed their effects on porcine embryonic development. Our results showed that the blastocyst formation rates of zygotes electroporated with gRNA3—alone and in combination—were significantly lower (p < 0.05) than those of zygotes electroporated with gRNA1. The mutation rates assessed by the PERV pol gene target site sequencing in individual blastocysts and pooled embryos at the 2-to-8-cell stage did not differ among the three gRNAs. However, the frequency of indel mutations in mutant embryos at the 2-to-8-cell stage trended higher in the embryos electroporated with gRNA3 alone and in combination. Embryonic development may be affected by gRNAs that induce high-frequency indel mutations.

Pigs with porcine endogenous retrovirus (PERV) inactivation are preferable donor sources for xenotransplantation because the PERV may act as an infectious pathogen for humans who receive pig organ xenotransplantation. However, inactivation of the PERV pol gene in porcine cells reportedly affects cell growth. The present study clarified the relationship between the mutation of the PERV pol gene in porcine embryos and their development. Three different gRNAs targeting the PERV pol gene were introduced into porcine zygotes by genome editing using electroporation of the Cas9 protein (GEEP) system. The results demonstrated a negative relationship between the embryonic development and the efficiency of target mutations in the PERV pol gene of the porcine embryos.

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