Comparison of the effects of introducing the CRISPR/Cas9 system by microinjection and electroporation into porcine embryos at different stages

Abstract Objective Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are used for generating genetically modified pigs. However, these methods create mosaic mutations in embryos. In this study, we evaluated whether the gene editing method and embryonic stage for gene editing affect the gene editing efficiency of porcine embryos. Results First, we designed five guide RNAs (gRNAs) targeting the B4GALNT2 gene and evaluated mutation efficiency by introducing each gRNA with Cas9 protein into zygotes by electroporation. Next, the optimized gRNA with Cas9 protein was introduced into 1-cell and 2-cell stage embryos by either microinjection or electroporation. The sequence of gRNA affected the bi-allelic mutation rate and mutation efficiency of blastocysts derived from electroporated embryos. Microinjection significantly decreased the cleavage rates in each embryonic stage and blastocyst formation rates in 2-cell stage embryos compared with electroporation (p