ABSTRACT. - Zygote microinjection is considered the most suitable technique to introduce CRISPR/Cas9 reagents for efficient genome editing in livestock. In this study, zygote electroporation was evaluated as an alternative to microinjection for CRISPR/Cas9-mediated genome editing in sheep. Four experiments were conducted on 3548 cumulusoocyte complexes. Acid Tyrode's solution (AT) was used to partially degrade the zona pellucida (ZP) to improve reagent entry, resulting in ZP thinning with longer AT exposure (P < 0.05). Although early embryo development was impaired by AT exposure (P < 0.05), blastocyst rates were similar across all groups by day 8. Electroporation conditions were optimized by testing pulse length (1 or 3 ms), with the best results from 6 pulses of 20 V for 3 ms with AT during 60 s. Electroporation with 500 ng/uL Cas9 and 300 ng/uL sgRNA with AT during 60 s achieved a 38.5 % mutation rate. When compared with conventional microinjection, electroporation had higher developmental rates but a lower mutation rate (21.4 % vs. 60.0 %; P < 0.05). These findings suggest that electroporation is a viable, cost-effective technique for genome editing in sheep. Nevertheless, further research will be required to fine-tune electroporation conditions and enhance efficiency in terms of mutation rate.
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