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oai:figshare.com:article/33962149 2026-09-22T05:38:38Z category_90 portal_316 item_type_3 month_year_09_2026

Supplementary file 1_Genome edited sheep generated by zygote electroporation of prime editor ribonucleoproteins.docx Zhixuan Yu (14103534) Junjin Li (16498668) Menghao Liu (20177988) Qi Qi (556563) Zengyi Duan (25087960) Ziyu Yang (9101302) Boyuan Guan (17743654) Jiawang Chen (9411900) Jian Hou (93442) Genetic Engineering genome editing prime editing ribonucleoprotein sheep zygote electroporation <p>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.</p> 2026-09-22T05:38:38Z Dataset Dataset 10.3389/fgeed.2026.1931608.s001 https://figshare.com/articles/dataset/Supplementary_file_1_Genome_edited_sheep_generated_by_zygote_electroporation_of_prime_editor_ribonucleoproteins_docx/33962149 CC BY 4.0