Genome edited sheep generated by zygote electroporation of prime editor ribonucleoproteins.

Prime editing (PE) enables precise genome modifications without double-strand breaks, yet its application in large mammals has been hindered by low efficiency and the technical bottlenecks of zygote microinjection. This study aimed to establish a scalable PE platform in sheep via the direct electroporation of a PE3 RNP formulation comprising PE2 protein, an epegRNA, and a nicking sgRNA into zygotes. We first validated functional PE2 protein and screened highly efficient enhanced pegRNAs (epegRNAs) targeting two high-value traits, including short-tail (TBXT) and prolificacy (FecB), in primary sheep fibroblasts, achieving editing efficiencies of up to 22.78%. Systematic optimization of zygote electroporation determined that 60 V combined with a PE2: pegRNA mass ratio of 3.5:1 maximized delivery efficiency, yielding gene editing efficiencies of 19.8% and 20.1% for TBXT and FecB, respectively, with no statistically significant differences detected in cleavage or blastocyst rates between electroporated and untreated zygotes. Following dual-target co-delivery, intended edits were detected at both the TBXT and FecB loci in separate preimplantation embryo subsets, with locus-specific editing rates comparable to those observed under single-target conditions. Following the transfer of 54 electroporated blastocysts, 14 live lambs were born. Targeted deep sequencing detected intended edits in five lambs (35.71%), including three at the FecB locus and two at the TBXT locus. Notably, a TBXT-edited lamb exhibiting a distinct short-tail phenotype (15 cm vs. 24.5 cm in wild-type) carried the highest allele frequency, reaching up to 50.97%, providing direct in vivo functional validation. Targeted analysis of predicted off-target sites detected no credible off-target editing, supporting the specificity of this transient RNP delivery strategy. Collectively, this RNP-electroporation framework provides a feasible and precise pathway for scalable prime editing in livestock, bypassing the limitations of conventional injection-based methods.