Genome mutation after the introduction of the gene editing by electroporation of Cas9 protein (GEEP) system into bovine putative zygotes.

Abstract

The present study was designed to investigate the effects of voltage strength on embryonic developmental rate and mutation efficiency in bovine putative zygotes during electroporation with the CRISPR/Cas9 system to target the MSTN gene at different time points after insemination. Results showed that there was no significant interaction between electroporation time and voltage strength on the embryonic cleavage and blastocyst formation rates. However, increasing the voltage strength to 20V/mm to electroporate the zygotes at 10h after the start of insemination yielded significantly lower blastocyst formation rates (P < 0.05) than those of the 10-V/mm electroporated zygotes. Mutation efficiency was then assessed in individual blastocysts by DNA sequence analysis of the target sites in the MSTN gene. A positive correlation between mutation rate and voltage strength was observed. The mutation efficiency in mutant blastocysts was significantly higher in the zygotes electroporated with 20V/mm at 10h after the start of insemination (P < 0.05) than in the zygotes electroporated at 15h, irrespective of the voltage strength. We also noted that a certain number of blastocysts from zygotes that were electroporated with more than 15V/mm at 10h (4.8-16.7%) and 20V/mm at 15h (4.8%) were biallelic mutants. Our results suggest that the voltage strength during electroporation as well as electroporation time certainly have effects on the embryonic developmental rate and mutation efficiency in bovine putative zygotes.