{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T06:04:56Z","timestamp":1790057096205,"version":"4.0.1"},"reference-count":34,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T00:00:00Z","timestamp":1790035200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Genome Ed."],"abstract":"<jats:p>\n                    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 (\n                    <jats:italic>TBXT<\/jats:italic>\n                    ) and prolificacy (\n                    <jats:italic>FecB<\/jats:italic>\n                    ), in primary sheep fibroblasts, achieving editing efficiencies of up to 22.78%. Systematic optimization of zygote electroporation determined that 60\u00a0V 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\n                    <jats:italic>TBXT<\/jats:italic>\n                    and\n                    <jats:italic>FecB<\/jats:italic>\n                    , 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\n                    <jats:italic>TBXT<\/jats:italic>\n                    and\n                    <jats:italic>FecB<\/jats:italic>\n                    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\n                    <jats:italic>FecB<\/jats:italic>\n                    locus and two at the\n                    <jats:italic>TBXT<\/jats:italic>\n                    locus. Notably, a\n                    <jats:italic>TBXT<\/jats:italic>\n                    -edited lamb exhibiting a distinct short-tail phenotype (15\u00a0cm vs. 24.5\u00a0cm in wild-type) carried the highest allele frequency, reaching up to 50.97%, providing direct\n                    <jats:italic>in vivo<\/jats:italic>\n                    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.\n                  <\/jats:p>","DOI":"10.3389\/fgeed.2026.1931608","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T05:40:42Z","timestamp":1790055642000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Genome edited sheep generated by zygote electroporation of prime editor ribonucleoproteins"],"prefix":"10.3389","volume":"8","author":[{"given":"Zhixuan","family":"Yu","sequence":"first","affiliation":[{"name":"State Key Laboratory of Animal Biotech Breeding and College of Biological Science, China Agricultural University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junjin","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Biotech Breeding and College of Biological Science, China Agricultural University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Menghao","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Biotech Breeding and College of Biological Science, China Agricultural University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qi","family":"Qi","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Biotech Breeding and College of Biological Science, China Agricultural University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zengyi","family":"Duan","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Biotech Breeding and College of Biological Science, China Agricultural University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziyu","family":"Yang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Biotech Breeding and College of Biological Science, China Agricultural University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Boyuan","family":"Guan","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Biotech Breeding and College of Biological Science, China Agricultural University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiawang","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Biotech Breeding and College of Biological Science, China Agricultural University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian","family":"Hou","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Biotech Breeding and College of Biological Science, China Agricultural University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,22]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"e2004832117","DOI":"10.1073\/pnas.2004832117","article-title":"Frequent loss of heterozygosity in CRISPR-Cas9\u2013edited early human embryos","volume":"118","author":"Alanis-Lobato","year":"2021","journal-title":"Proc. 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