Zona pellucida treatment before CRISPR/Cas9‐mediated genome editing of porcine zygotes

BACKGROUND: Increasing the permeability of the zona pellucida (ZP) of oocytes before CRISPR/Cas9 electroporation may improve the efficiency of gene editing; however, the effects of this approach on subsequent developmental processes are unclear. In this study, the effects of ZP treatment before electroporation on embryonic development and gene editing in porcine embryos were evaluated. METHODS: The ZP of zygotes was weakened or removed by exposure to 0.5% actinase E, followed by electroporation of the Cas9 protein with guide RNA targeting GGTA1. RESULTS: The blastocyst formation rate of ZP‐free zygotes after electroporation was significantly lower (p < 0.05) than that of ZP‐intact zygotes. The mutation rate in blastocysts from ZP‐weakened zygotes was similar to that in ZP‐intact zygotes, whereas ZP removal increased the mutation rate. The mutation efficiency in blastocysts from electroporated zygotes did not differ among ZP treatment groups. CONCLUSIONS: Our results indicate that weakening the ZP does not affect the developmental competence, mutation rate, or mutation efficiency of electroporated zygotes, whereas ZP removal has a detrimental effect on embryonic development but may increase the mutation rate.