Supplementary file 1_Genome edited sheep generated by zygote electroporation of prime editor ribonucleoproteins.docx

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.