Data availabilityHigh-throughput sequencing data generated by this study have been deposited in the GEO database under accession GSE250497. Additional supporting data can be found in Supplementary Information or from the corresponding author upon reasonable request. Source data are provided with this paper.ReferencesLi, W. et al. Androgenetic haploid embryonic stem cells produce live transgenic mice. Nature 490, 407–411 (2012).Article CAS PubMed Google Scholar Yang, H. et al. Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells. Cell 149, 605–617 (2012).Article CAS PubMed Google Scholar Li, W. et al. Genetic modification and screening in rat using haploid embryonic stem cells. Cell Stem Cell 14, 404–414 (2014).Article CAS PubMed Google Scholar Lin, J. C. & Van Eenennaam, A. L. Electroporation-mediated genome editing of livestock zygotes. Front. 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This study was supported by the National Natural Science Foundation of China (32341052 to L.Y., 32360837 to L.Y. and 32488101 to S.G.), Scientific and Technological Innovation 2030 (2023ZD0404803 to L.Y.), Inner Mongolia Open Competition Projects (2022JBGS0025 to L.Y.), Inner Mongolia Science and Technology Leading Team (2022LJRC0006 to G.L.), Inner Mongolia Science and Technology Major Projects (2022ZD0008 to L.Y., 2023KJHZ0028 to L.Y. and 2025KYPT0101 to L.Y.), Inner Mongolia Young Talents Projects (NJYT23138 to L.Y.), Inner Mongolia Natural Science Foundation (2023MS03004 to L.Y.), National Agricultural Science and Technology Project (NK2022130203 to L.Y.), Collaborative Innovation among Universities in Hohhot (XTCX202306 to L.Y.), Ministry of Education Engineering Centre Project (JYBGCSYS2022 to L.Y.), Xinjiang Uygur Science and Technology Major Project (2023A0201116 to L.Y.) and TongLiao Open Competition Projects (TL2024TW0020103 to L.Y.).Author informationAuthor notesDeceased: Li Zhang.These authors contributed equally: Lei Yang, Anqi Di, Lishuang Song.These authors jointly supervised this work: Lei Yang, Shaorong Gao, Guangpeng Li.Authors and AffiliationsCollege of Life Sciences, Inner Mongolia University, Hohhot, ChinaLei Yang, Anqi Di, Lishuang Song, Xuefei Liu, Di Wu, Song Wang, Zhenting Hao, Lige Bu, Chunling Bai, Guanghua Su, Zhuying Wei, Li Zhang & Guangpeng LiFrontier Science Center for Stem Cell Research, School of Life Sciences and Technology, Tongji University, Shanghai, ChinaLei Yang & Shaorong GaoKey Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, ChinaSong Wang & Zhonghua LiuAuthorsLei YangView author publicationsSearch author on:PubMed Google ScholarAnqi DiView author publicationsSearch author on:PubMed Google ScholarLishuang SongView author publicationsSearch author on:PubMed Google ScholarXuefei LiuView author publicationsSearch author on:PubMed Google ScholarDi WuView author publicationsSearch author on:PubMed Google ScholarSong WangView author publicationsSearch author on:PubMed Google ScholarZhenting HaoView author publicationsSearch author on:PubMed Google ScholarLige BuView author publicationsSearch author on:PubMed Google ScholarChunling BaiView author publicationsSearch author on:PubMed Google ScholarGuanghua SuView author publicationsSearch author on:PubMed Google ScholarZhuying WeiView author publicationsSearch author on:PubMed Google ScholarLi ZhangView author publicationsSearch author on:PubMed Google ScholarZhonghua LiuView author publicationsSearch author on:PubMed Google ScholarShaorong GaoView author publicationsSearch author on:PubMed Google ScholarGuangpeng LiView author publicationsSearch author on:PubMed Google ScholarContributionsL.Y., A.D., X.L., L.S., D.W., S.W., Z.H., L.B., C.B., G.S., Z.W. and L.Z. performed experiments. L.Y., G.L. and S.G. designed experiments. L.Y. and G.L. wrote the manuscript.Corresponding authorsCorrespondence to Lei Yang, Shaorong Gao or Guangpeng Li.Ethics declarationsCompeting interestsThe authors declare no competing interests.Peer reviewPeer review informationNature Biotechnology thanks Björn Oback, Steven Stice and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Generation of ruminant haploid androgenetic embryos.a. Schematic overview of haSCs derivation, haploid embryos were generated by removing female pronucleus from normal diploid zygotes. ♂, male pronucleus; ♀, female pronucleus. b. Immunostaining for 5-hydroxymethylcytosine (5hmC) in bovine (left) and ovine (right) embryos. The haploid androgenetic embryos are generated by sperm injection into enucleated oocytes or by removing female pronucleus from fertilized oocytes. The diploid IVF embryos were used as controls. ♂, male pronucleus; ♀, female pronucleus; n = 3 independent experiments with similar results. Scale bar, 25 μm. c. Immunostaining for trophectoderm marker CDX2 in the bovine and ovine blastocysts on day 7 after activation or insemination. The haploid and diploid embryos are generated as described in Extended Data Fig. 1b. n = 3 independent experiments with similar results. Scale bar, 50 μm. d. Quantification for the cell numbers in the bovine and ovine blastocysts on day 7 after activation or insemination. The haploid and diploid embryos are generated as described in Extended Data Fig. 1b; ICM, inner cell mass; Data are mean ± s.d. (n = 18 IVF, n = 19 injecting, n = 21 removing (top left); n = 18 IVF, n = 19 injecting, n = 21 removing (top right); n = 22 IVF, n = 19 injecting, n = 23 removing (bottom left); n represents total embryos of three independent experiments); P values are from unpaired, two-tailed Student’s t-tests.Extended Data Fig. 2 Derivation of ruminant haSCs from haploid androgenetic embryos.a. List of main components in the well-known medium that support the derivation of mouse, human, and bovine diploid SCs, including 2i/LIF, mTeSR1, t2iL+Gö, 5i/L/A, ABCL, LCDM, EPSC, CTFR, and bEPSCM. The brief names of these chemicals and their respective pathways which they regulate are also shown. b. Morphology of bovine haploid androgenetic ICMs under different culture medium conditions at indicated time points. D, day; P, passage; n = 3 independent experiments with similar results. Scale bars, 200 μm. c. Representative immunofluorescence (IF) images of pluripotency factors in the indicated primary outgrowth. n = 3 independent experiments with similar results. Scale bar, 100 μm. d, e. The efficiency of outgrowth and haSCs derivation of haploid androgenetic ICMs by different culture mediums. n = 3 independent experiments with similar results. N = total number of ICMs used for each condition.Extended Data Fig. 3 FACE medium supports ruminant haSCs long-term culture.a. List of different combinations of small molecules used in this study. b. Summary of b-haSCs derivation efficiency from bovine haploid androgenetic ICMs by different combinations of small molecules. c. Morphology of bovine haploid androgenetic ICMs under different combinations of small molecules. Scale bars, 200 μm. d. The total cell numbers of b-haSCs during cell passaging under different concentrations of Activin-A. Cells were plated at 5 ×105 cells per well and were cultured for 4 days; Data are mean ± s.d. (n = 3 independent experiments); P values are from unpaired, two-tailed Student’s t-tests. e. Haploidy analysis in b-haSCs derived using 10 ng/mL and 20 ng/mL of Activin-A. The figure on the left is the same as Fig. 1e; n = 3 independent experiments with similar results. Note that 10 ng/mL Activin-A was beneficial for maintaining haploidy stability.Extended Data Fig. 4 Further characterization of ruminant haSCs.a. DNA copy number variation (CNV) analysis of ruminant haSCs at passage 25. Note that no significant genomic alternations. The results were displayed on a log2 scale. Chromosomes are arranged in numerical order and in different colours. b. Summary of karyotyping analysis of ruminant haSCs at different passages. Total, the total number of examined cells in which chromosome was successfully spread. c. Determination of the sex chromosome of ruminant haSCs by PCR assay. The primers are specific for X chromosome-specific PHEX and Y chromosome-specific ZFY genes. Normal XX and XY diploid SCs were used as controls. M, DNA marker. n = 3 independent experiments with similar results. d. Scatter-plots showing the reproducibility of buRNA-seq between different biological replicates of ruminant haSCs. The Pearson’s correlation coefficients (Cor.) are shown. e. Principal-component analysis (PCA) showing the separation of b-haSCs and bovine pre-implantation embryos. f. Scatter plot based on PCA of b-haSCs, diploid primed b-SCs, and diploid expanded b-SCs, showing that the projection position of b-haSCs is located between expanded and primed b-SCs. g. Correlation matrices showing coefficients among b-haSCs, diploid primed b-SCs, diploid expanded b-SCs, and pre-implantation bovine embryos. h. Heatmap displaying the differentially expressed genes (DEGs) among b-haSCs, primed b-SCs, and expanded b-SCs (P 2, FPKM > 2) in growth-retarded Pro-iCHI fetuses compared with normal Pro-iCHI fetuses and control IVF fetuses. FC, fold change; FPKM, fragments per kilobase of transcript per million mapped reads. e. Bisulfite pyrosequencing analysis of paternally imprinted H19 DMR in bovine and ovine fetuses produced by Pro-iCHI or IVF approach. Note that all the fetuses maintained allelic-biased DNA methylation in H19 DMR. The filled and open squares represent methylated and unmethylated CpG sites, respectively. f. Schematic representation of the ePE vector used in this study. This “all-in-one” episomal plasmid contained the necessary elements of CRISPR-prime editor (PE), including Cas9-nickase and reverse-transcriptase (RT) fusion sequence, and a prime-editing guide RNA (pegRNA). The vector also contains an EF1a promoter-driven EGFP for tracking transfection efficiency. Puro, Puromycin resistance gene. g. Western blots analysis of MSTN protein in wild-type (WT) and MSTN-edited ruminant haSCs. n = 3 independent experiments with similar results. h. Episomal plasmid was decreased within haSCs over time after withdrawing puromycin drug selection. Numbers in the X-axis indicate cell passage number (passage cells every 5 days). Amp, ampicillin; Actin, beta-actin; mean ± s.d., n = 3 independent experiments; amplification cycle was maintained at 40 and the cycle threshold (Ct) values > 35 were considered as not detected (ND). i. Western blots analysis of MSTN protein in wild-type (WT) and MSTN-edited live cattle. n = 3 independent experiments with similar results. j. Sanger sequencing results of the targeting site in wild-type (WT) and MSTN-edited live cattle, the deletion sizes (Δ) are indicated. k. Genomic PCR showing the absence of exo-vector in lamb and calf produced by Pro-iCHI::ePE system. The positive and negative controls were amplified from the episomal plasmid and water, respectively. n = 3 independent experiments with similar results. Lane 1: positive control; Lane 2: sheep; Lanes 3-5: cattle; M, DNA marker.Source dataSupplementary informationSupplementary InformationSupplementary Figs. 1–8, Discussion and Tables 1–8.Reporting SummarySupplementary Data 1Unprocessed gel for Supplementary Fig. 1b,d.Supplementary Data 2Unprocessed gel for Supplementary Fig. 7d.Source dataSource Data Extended Data Fig. 4Unprocessed gels for Extended Data Fig. 4c.Source Data Extended Data Fig. 5Unprocessed gels and western blot for Extended Data Fig. 5d,e.Source Data Extended Data Fig. 6Unprocessed gels for Extended Data Fig. 6b.Source Data Extended Data Fig. 10Unprocessed gels and western blot for Extended Data Fig. 10g,i,k.Rights and permissionsSpringer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.Reprints and permissionsAbout this article