Zygote origin shapes sensitivity to electrical voltage field and editing susceptibility after CRISPR/Cas9-RNP electroporation in bovine embryos.

Despite major progress in generation of edited embryos and offspring via CRISPR/Cas9-RNP electroporation, editing outcomes remain highly variable. This variability may be due to intrinsic differences between in vivo-derived (IVV) and in vitro-derived (IVT) zygotes, which may play a more decisive role than previously anticipated. Therefore, this study aimed to determine how zygote origin shapes the sensitivity to electrical voltage field and editing susceptibility after CRISPR/Cas9-RNP electroporation in the bovine model. The present study systematically compared subsequent developmental competence and editing events in IVV- and IVT-derived bovine zygotes across defined electroporation voltages and pulse durations. Notably, CRISPR/Cas9-RNP electroporation of IVV-derived bovine zygotes of comparable age and collected by tubal embryo flushing (10-18 h post ovulation, hpo) resulted in markedly lower survival rates at 25 and 30 V compared to 20 V. In contrast, higher (p < 0.05) editing frequencies were observed at 25 V and 30 V. Clear differences, however, were observed between the two zygote groups. Following CRISPR/Cas9 RNP electroporation, IVV-derived zygotes yielded a higher proportion of unedited blastocysts (p < 0.05), absence of full edited embryos, a significantly increased frequency of monoallelic edits and reduced rates of mosaicism compared to IVT-derived zygotes. Collectively, the results of the present study clearly underscore a significant lower editing efficiency to pulse sensitivity ratio in IVV-derived zygotes compared to their IVT counterparts. To our knowledge, this is the first study to use bovine IVV-derived zygotes for genome editing using RNP-electroporation to elucidate how zygote origin shapes sensitivity to electrical voltage field and editing efficiency.