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Background and Aim: We previously developed the gene-editing by electroporation (EP) of Cas9 protein method, in which the CRISPR/Cas9 system was introduced into porcine in vitro fertilized (IVF) zygotes through EP to disrupt a target gene. This method should be further developed, and a combination of EP and MI methods should be evaluated in pigs. This study aimed to determine that a combination of microinjection (MI) and EP of CRISPR/Cas9 system could increase the rates of biallelic mutation for triple-gene knockout in porcine blastocysts. We targeted the pancreatic and duodenal homeobox1 (PDX1) gene using cytoplasmic MI 1 h before or after EP, which was used to edit alpha-1,3-galactosyltransferase (GGTA1) and cytidine 32 monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes in porcine zygotes.Materials and Methods: We introduced guide RNAs targeting PDX1, GGTA1, and CMAH with the Cas9 protein into IVF zygotes (one-cell stage) through EP 10 h after the start of IVF (IVF; EP group) or in combination with MI (1 h before, MI-EP group, or after EP treatment EP-MI group) and evaluated the blastocyst formation rate and efficiency of target mutations in the resulting blastocysts.Results: Our results revealed a significant reduction in the rate of blastocyst formation in the two groups that underwent MI before and after EP (MI-EP and EP-MI group), compared with that in the groups treated with EP alone (EP group) (p=0.0224 and p&lt;0.0001, respectively) and control (p=0.0029 and p&lt;0.0001, respectively). There was no significant difference in the total mutation rates among the treatment groups in the resulting blastocysts. As an only positive effect of additional MI treatment, the rate of blastocysts carrying biallelic mutations in at least one target gene was higher in the MI-EP group than in the EP group. However, there was no difference in the rates of embryos carrying biallelic mutations in more than 2 target genes.Conclusion: These results indicate that although a combination of MI and EP does not improve the mutation efficiency or biallelic mutation for triple-gene knockout, MI treatment before EP is better to reduce mortality in porcine zygotic gene editing through a combination of MI and EP.

Triple gene editing in porcine embryos using electroporation alone or in combination with microinjection