{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T16:43:13Z","timestamp":1790095393218,"version":"4.0.1"},"reference-count":40,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2024,8,23]],"date-time":"2024-08-23T00:00:00Z","timestamp":1724371200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Startup Foundation for Advanced Talents of Shihezi university","award":["RCZK2021B34"],"award-info":[{"award-number":["RCZK2021B34"]}]},{"name":"Startup Foundation for Advanced Talents of Shihezi university","award":["NYHXGG2023AA101"],"award-info":[{"award-number":["NYHXGG2023AA101"]}]},{"name":"Startup Foundation for Advanced Talents of Shihezi university","award":["KJTP202401"],"award-info":[{"award-number":["KJTP202401"]}]},{"name":"Startup Foundation for Advanced Talents of Shihezi university","award":["SRP2024014"],"award-info":[{"award-number":["SRP2024014"]}]},{"name":"Bingtuan Science and Technology Project","award":["RCZK2021B34"],"award-info":[{"award-number":["RCZK2021B34"]}]},{"name":"Bingtuan Science and Technology Project","award":["NYHXGG2023AA101"],"award-info":[{"award-number":["NYHXGG2023AA101"]}]},{"name":"Bingtuan Science and Technology Project","award":["KJTP202401"],"award-info":[{"award-number":["KJTP202401"]}]},{"name":"Bingtuan Science and Technology Project","award":["SRP2024014"],"award-info":[{"award-number":["SRP2024014"]}]},{"name":"Science and Technology Specialist Team Service Program of Shihezi University","award":["RCZK2021B34"],"award-info":[{"award-number":["RCZK2021B34"]}]},{"name":"Science and Technology Specialist Team Service Program of Shihezi University","award":["NYHXGG2023AA101"],"award-info":[{"award-number":["NYHXGG2023AA101"]}]},{"name":"Science and Technology Specialist Team Service Program of Shihezi University","award":["KJTP202401"],"award-info":[{"award-number":["KJTP202401"]}]},{"name":"Science and Technology Specialist Team Service Program of Shihezi University","award":["SRP2024014"],"award-info":[{"award-number":["SRP2024014"]}]},{"name":"Research Training Program for Undergraduates of Shihezi University","award":["RCZK2021B34"],"award-info":[{"award-number":["RCZK2021B34"]}]},{"name":"Research Training Program for Undergraduates of Shihezi University","award":["NYHXGG2023AA101"],"award-info":[{"award-number":["NYHXGG2023AA101"]}]},{"name":"Research Training Program for Undergraduates of Shihezi University","award":["KJTP202401"],"award-info":[{"award-number":["KJTP202401"]}]},{"name":"Research Training Program for Undergraduates of Shihezi University","award":["SRP2024014"],"award-info":[{"award-number":["SRP2024014"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The utilization of electroporation for delivering CRISPR\/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9\/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.<\/jats:p>","DOI":"10.3390\/ijms25179145","type":"journal-article","created":{"date-parts":[[2024,8,26]],"date-time":"2024-08-26T05:16:24Z","timestamp":1724649384000},"page":"9145","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Electroporation Delivery of Cas9 sgRNA Ribonucleoprotein-Mediated Genome Editing in Sheep IVF Zygotes"],"prefix":"10.3390","volume":"25","author":[{"given":"Wenhui","family":"Pi","sequence":"first","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University, Shihezi 832003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guangyu","family":"Feng","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University, Shihezi 832003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minghui","family":"Liu","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University, Shihezi 832003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3320-2840","authenticated-orcid":false,"given":"Cunxi","family":"Nie","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University, Shihezi 832003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0242-9332","authenticated-orcid":false,"given":"Cheng","family":"Chen","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University, Shihezi 832003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingjing","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Limin","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pengcheng","family":"Wan","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Changbin","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ping","family":"Zhou","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,8,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"545","DOI":"10.1007\/s00335-023-10018-z","article-title":"Recent advances in the genomic resources for sheep","volume":"34","author":"Woolley","year":"2023","journal-title":"Mamm. Genome"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Kalds, P., Zhou, S., Cai, B., Liu, J., Wang, Y., Petersen, B., Sonstegard, T., Wang, X., and Chen, Y. (2019). Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era. Front. Genet., 10.","DOI":"10.3389\/fgene.2019.00750"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"eadd8643","DOI":"10.1126\/science.add8643","article-title":"CRISPR technology: A decade of genome editing is only the beginning","volume":"379","author":"Wang","year":"2023","journal-title":"Science"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Wang, X., Niu, Y., Zhou, J., Yu, H., Kou, Q., Lei, A., Zhao, X., Yan, H., Cai, B., and Shen, Q. (2016). Multiplex gene editing via CRISPR\/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep. Sci. Rep., 6.","DOI":"10.1038\/srep32271"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Crispo, M., Mulet, A.P., Tesson, L., Barrera, N., Cuadro, F., dos Santos-Neto, P.C., Nguyen, T.H., Cr\u00e9n\u00e9guy, A., Brusselle, L., and Aneg\u00f3n, I. (2015). Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR\/Cas9 Technology and Microinjection into Zygotes. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0136690"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Zhang, X., Li, W., Liu, C., Peng, X., Lin, J., He, S., Li, X., Han, B., Zhang, N., and Wu, Y. (2017). Alteration of sheep coat color pattern by disruption of ASIP gene via CRISPR Cas9. Sci. Rep., 7.","DOI":"10.1038\/s41598-017-08636-0"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Menchaca, A., Dos Santos-Neto, P.C., Souza-Neves, M., Cuadro, F., Mulet, A.P., Tesson, L., Chenouard, V., Guiff\u00e8s, A., Heslan, J.M., and Gantier, M. (2020). Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair. Sci. Rep., 10.","DOI":"10.1038\/s41598-020-62879-y"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Lin, Y., Li, J., Li, C., Tu, Z., Li, S., Li, X.J., and Yan, S. (2022). Application of CRISPR\/Cas9 System in Establishing Large Animal Models. Front. Cell Dev. Biol., 10.","DOI":"10.3389\/fcell.2022.919155"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1253","DOI":"10.1038\/nprot.2018.012","article-title":"Efficient mouse genome engineering by CRISPR-EZ technology","volume":"13","author":"Modzelewski","year":"2018","journal-title":"Nat. Protoc."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Lin, J.C., and Van Eenennaam, A.L. (2021). Electroporation-Mediated Genome Editing of Livestock Zygotes. Front. Genet., 12.","DOI":"10.3389\/fgene.2021.648482"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Kaneko, T., Sakuma, T., Yamamoto, T., and Mashimo, T. (2014). Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Sci. Rep., 4.","DOI":"10.1038\/srep06382"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1534\/genetics.115.176594","article-title":"Efficient CRISPR\/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease","volume":"200","author":"Qin","year":"2015","journal-title":"Genetics"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1016\/j.jgg.2016.02.004","article-title":"Delivery of Cas9 Protein into Mouse Zygotes through a Series of Electroporation Dramatically Increases the Efficiency of Model Creation","volume":"43","author":"Wang","year":"2016","journal-title":"J. Genet. Genom."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"24195","DOI":"10.1073\/pnas.2010102117","article-title":"Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males","volume":"117","author":"Ciccarelli","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Tanihara, F., Hirata, M., Nguyen, N.T., Le, Q.A., Hirano, T., Takemoto, T., Nakai, M., Fuchimoto, D.I., and Otoi, T. (2018). Generation of a TP53-modified porcine cancer model by CRISPR\/Cas9-mediated gene modification in porcine zygotes via electroporation. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0206360"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Sundby, L.J., Southern, W.M., Hawbaker, K.M., Trujillo, J.M., Perrin, B.J., and Ervasti, J.M. (2022). Nucleotide- and Protein-Dependent Functions of Actg1. Mol. Biol. Cell, 33.","DOI":"10.1091\/mbc.E22-02-0054"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1038\/387083a0","article-title":"Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member","volume":"387","author":"McPherron","year":"1997","journal-title":"Nature"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1093\/nsr\/nwz013","article-title":"Genome editing in large animals: Current status and future prospects","volume":"6","author":"Zhao","year":"2019","journal-title":"Natl. Sci. Rev."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Zhou, S., Kalds, P., Luo, Q., Sun, K., Zhao, X., Gao, Y., Cai, B., Huang, S., Kou, Q., and Petersen, B. (2022). Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality. BMC genomics, 23.","DOI":"10.1186\/s12864-022-08594-6"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Guo, R., Wang, H., Meng, C., Gui, H., Li, Y., Chen, F., Zhang, C., Zhang, H., Ding, Q., and Zhang, J. (2023). Efficient and Specific Generation of MSTN-Edited Hu Sheep Using C-CRISPR. Genes, 14.","DOI":"10.3390\/genes14061216"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Mahdi, A.K., Medrano, J.F., and Ross, P.J. (2022). Single-Step Genome Editing of Small Ruminant Embryos by Electroporation. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms231810218"},{"key":"ref_22","first-page":"328","article-title":"Optimizing electroporation conditions in primary and other difficult-to-transfect cells","volume":"19","author":"Jordan","year":"2008","journal-title":"J. Biomol. Tech."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1111\/rda.13106","article-title":"Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos","volume":"53","author":"Nishio","year":"2018","journal-title":"Reprod. Domest. Anim."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Anzalone, D.A., Palazzese, L., Czernik, M., Sabatucci, A., Valbonetti, L., Capra, E., and Loi, P. (2021). Controlled spermatozoa-oocyte interaction improves embryo quality in sheep. Sci. Rep., 11.","DOI":"10.1038\/s41598-021-02000-z"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., and Murray, J.D. (2020). Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Sci. Rep., 10.","DOI":"10.1038\/s41598-020-78264-8"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Miskel, D., Poirier, M., Beunink, L., Rings, F., Held, E., Tholen, E., Tesfaye, D., Schellander, K., Salilew-Wondim, D., and Blaschka, C. (2022). The cell cycle stage of bovine zygotes electroporated with CRISPR\/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events. Sci. Rep., 12.","DOI":"10.1038\/s41598-022-25117-1"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Vilarino, M., Rashid, S.T., Suchy, F.P., McNabb, B.R., van der Meulen, T., Fine, E.J., Ahsan, S.D., Mursaliyev, N., Sebastiano, V., and Diab, S.S. (2017). CRISPR\/Cas9 microinjection in oocytes disables pancreas development in sheep. Sci. Rep., 7.","DOI":"10.1038\/s41598-017-17805-0"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"598","DOI":"10.1007\/s11626-019-00385-w","article-title":"Genome mutation after the introduction of the gene editing by electroporation of Cas9 protein (GEEP) system into bovine putative zygotes","volume":"55","author":"Namula","year":"2019","journal-title":"Vitro Cell Dev. Biol. Anim."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Qiao, J., Li, W., Lin, S., Sun, W., Ma, L., and Liu, Y. (2019). Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins. Commun. Biol., 2.","DOI":"10.1038\/s42003-019-0402-x"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"985","DOI":"10.1016\/j.stemcr.2021.02.013","article-title":"Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9\/sgRNA Ribonucleoprotein","volume":"16","author":"Kagita","year":"2021","journal-title":"Stem Cell Rep."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1038\/nbt.2647","article-title":"DNA targeting specificity of RNA-guided Cas9 nucleases","volume":"31","author":"Hsu","year":"2013","journal-title":"Nat. Biotechnol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e00471","DOI":"10.7554\/eLife.00471","article-title":"RNA-programmed genome editing in human cells","volume":"2","author":"Jinek","year":"2013","journal-title":"eLife"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2837","DOI":"10.1093\/nar\/gkw138","article-title":"Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3\u2032-terminal segment of guide RNA","volume":"44","author":"Mekler","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Tanihara, F., Hirata, M., Namula, Z., Do, L.T.K., Yoshimura, N., Lin, Q., Takebayashi, K., Sakuma, T., Yamamoto, T., and Otoi, T. (2023). Pigs with an INS point mutation derived from zygotes electroporated with CRISPR\/Cas9 and ssODN. Front. Cell Dev. Biol., 11.","DOI":"10.3389\/fcell.2023.884340"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Remy, S., Chenouard, V., Tesson, L., Usal, C., M\u00e9noret, S., Brusselle, L., Heslan, J.M., Nguyen, T.H., Bellien, J., and Merot, J. (2017). Generation of gene-edited rats by delivery of CRISPR\/Cas9 protein and donor DNA into intact zygotes using electroporation. Sci. Rep., 7.","DOI":"10.1038\/s41598-017-16328-y"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1038\/s41467-017-02706-7","article-title":"Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses","volume":"9","author":"Yoon","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"286","DOI":"10.1016\/j.isci.2018.10.030","article-title":"Intra-embryo Gene Cassette Knockin by CRISPR\/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector","volume":"9","author":"Mizuno","year":"2018","journal-title":"iScience"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3780","DOI":"10.1016\/j.celrep.2019.05.103","article-title":"CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection","volume":"27","author":"Chen","year":"2019","journal-title":"Cell Rep."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"101924","DOI":"10.1016\/j.xpro.2022.101924","article-title":"An updated protocol for in vitro bovine embryo production","volume":"4","author":"Speckhart","year":"2023","journal-title":"STAR Protoc."},{"key":"ref_40","first-page":"1747","article-title":"Use of the CRISPR-Cas9 system in sheep fibroblasts to lead fluorescent tags into ACTG1 gene","volume":"58","author":"Guo","year":"2021","journal-title":"Xinjiang Agric. Sci."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/17\/9145\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T15:42:02Z","timestamp":1760110922000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/17\/9145"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,8,23]]},"references-count":40,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2024,9]]}},"alternative-id":["ijms25179145"],"URL":"https:\/\/doi.org\/10.3390\/ijms25179145","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,8,23]]}}}