Abstract
ObjectiveCytoplasmic 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.ResultsFirst, 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 < 0.05). However, the bi-allelic mutation rate and mutation efficiency of blastocysts from the 1-cell stage embryos edited using microinjection were significantly higher (p < 0.05) than those of blastocysts from the 2-cell stage embryos edited by both methods. These results indicate that the gene editing method and embryonic stage for gene editing may affect the genotype and mutation efficiency of the resulting embryos.
Highlights
Modified pigs are expected to be an excellent disease model contributing to human medicine [1, 2] and to be ideal organ donors for human xenotransplantation [3, 4]
There were no significant differences in the cleavage and blastocyst formation rates of embryos edited by electroporation among the different guide RNAs (gRNAs) groups (Fig. 1a, b)
The blastocyst formation rate of 1-cell stage and 2-cell stage embryos having Cas9 and gRNA introduced via microinjection was significantly lower (p < 0.05) than that of 2-cell stage embryos having gRNA and Cas9 introduced via electroporation
Summary
Experiment 1 Representative images of the genotyping are shown in Additional file 3: Figure S1. The mutation efficiency in gene-edited blastocysts derived from embryos electroporated with gRNA #1 significantly increased (p < 0.05) compared with that with gRNAs #2 and #3 (Fig. 1d). Experiment 2 the cleavage rates of embryos treated using the microinjection method significantly decreased compared with those of embryos treated using the electroporation method, irrespective of the embryonic stage (p < 0.05). When the embryonic stage and gene editing method were same, the cleavage rates and blastocyst formation rates of embryos treated with gRNA and Cas were statistically same as that of embryos treated without gRNA and Cas. The total mutation rate of blastocysts derived from the 2-cell stage embryos edited using the microinjection method significantly decreased (p < 0.05) compared with that of the other treatment groups (Fig. 2a). The rates of bi-allelic mutant and mutation efficiency of blastocysts from the 1-cell stage embryos edited using the microinjection method were significantly higher (p < 0.05) than those of blastocysts from the 2-cell stage embryos edited using both methods (Fig. 2a, b)
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