{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T18:39:18Z","timestamp":1785436758390,"version":"3.56.0"},"reference-count":35,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2026,6,20]],"date-time":"2026-06-20T00:00:00Z","timestamp":1781913600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Guangdong Provincial Key Research and Development Program","award":["2022B0202090003"],"award-info":[{"award-number":["2022B0202090003"]}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"crossref","award":["JP21KK0124"],"award-info":[{"award-number":["JP21KK0124"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"crossref","award":["JP22K19896"],"award-info":[{"award-number":["JP22K19896"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"crossref","award":["JP23K23764"],"award-info":[{"award-number":["JP23K23764"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"crossref","award":["JP23K23805"],"award-info":[{"award-number":["JP23K23805"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"crossref"}]},{"award":["JP21KK0124"],"award-info":[{"award-number":["JP21KK0124"]}],"id":[{"id":"https:\/\/ror.org\/00hhkn466","id-type":"ROR","asserted-by":"publisher"}]},{"award":["JP22K19896"],"award-info":[{"award-number":["JP22K19896"]}],"id":[{"id":"https:\/\/ror.org\/00hhkn466","id-type":"ROR","asserted-by":"publisher"}]},{"award":["JP23K23764"],"award-info":[{"award-number":["JP23K23764"]}],"id":[{"id":"https:\/\/ror.org\/00hhkn466","id-type":"ROR","asserted-by":"publisher"}]},{"award":["JP23K23805"],"award-info":[{"award-number":["JP23K23805"]}],"id":[{"id":"https:\/\/ror.org\/00hhkn466","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>The CRISPR\/Cas9 system has been widely used for gene editing in various species; however, mosaicism remains a significant challenge. This study aimed to improve gene editing efficiency and reduce mosaicism in porcine embryos by exploring double electroporation pre- and post-in vitro fertilization combined with zona pellucida (ZP) removal. We evaluated the effects of these treatments on the development and mutation rates of oocytes\/zygotes edited with guide RNAs (gRNAs) targeting GGTA1, CMAH, or B4GALNT2 genes. Double electroporation significantly increased the total and biallelic mutation rates in ZP-intact zygotes but not in ZP-free zygotes edited using GGTA1-targeted gRNAs. All blastocysts from ZP-free zygotes exhibited biallelic mutations following double electroporation. For the CMAH gene, all blastocysts exhibited mutations (biallelic mutations \u2265 80%); however, double electroporation and ZP removal did not affect their mutation rates or efficiency. For the B4GALNT2 gene, double electroporation significantly increased total mutation rates in ZP-intact zygotes, whereas all blastocysts from ZP-free zygotes showed biallelic mutation. These findings suggest that double electroporation, particularly with ZP removal, may enhance gene-editing efficiency, reduce mosaicism and improve the success of genetic modifications.<\/jats:p>","DOI":"10.3390\/ani16121919","type":"journal-article","created":{"date-parts":[[2026,6,22]],"date-time":"2026-06-22T09:49:55Z","timestamp":1782121795000},"page":"1919","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Double Electroporation Combined with Zona Pellucida Removal Improves Biallelic Genome Editing Efficiency in Porcine Embryos"],"prefix":"10.3390","volume":"16","author":[{"given":"Nanaka","family":"Torigoe","sequence":"first","affiliation":[{"name":"College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524088, China"},{"name":"Bio-Innovation Research Center, Tokushima University, Tokushima 7793233, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0382-959X","authenticated-orcid":false,"given":"Takeshige","family":"Otoi","sequence":"additional","affiliation":[{"name":"College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524088, China"},{"name":"Bio-Innovation Research Center, Tokushima University, Tokushima 7793233, Japan"},{"name":"Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya 60115, Indonesia"},{"name":"Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1646-0581","authenticated-orcid":false,"given":"Manita","family":"Wittayarat","sequence":"additional","affiliation":[{"name":"Faculty of Veterinary Science, Prince of Songkla University, Songkhla 90110, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0073-1394","authenticated-orcid":false,"given":"Oky Setyo","family":"Widodo","sequence":"additional","affiliation":[{"name":"Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya 60115, Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0225-5854","authenticated-orcid":false,"given":"Theerawat","family":"Tharasanit","sequence":"additional","affiliation":[{"name":"Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9470-8426","authenticated-orcid":false,"given":"Kaywalee","family":"Chatdarong","sequence":"additional","affiliation":[{"name":"Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-7045-3922","authenticated-orcid":false,"given":"Megumi","family":"Nagahara","sequence":"additional","affiliation":[{"name":"College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524088, China"},{"name":"Bio-Innovation Research Center, Tokushima University, Tokushima 7793233, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4673-1581","authenticated-orcid":false,"given":"Maki","family":"Hirata","sequence":"additional","affiliation":[{"name":"College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524088, China"},{"name":"Bio-Innovation Research Center, Tokushima University, Tokushima 7793233, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fuminori","family":"Tanihara","sequence":"additional","affiliation":[{"name":"College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524088, China"},{"name":"Bio-Innovation Research Center, Tokushima University, Tokushima 7793233, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhao","family":"Namula","sequence":"additional","affiliation":[{"name":"College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524088, China"},{"name":"Bio-Innovation Research Center, Tokushima University, Tokushima 7793233, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1462","DOI":"10.1089\/dna.2020.6474","article-title":"CRISPR\/Cas9-mediated gene editing in porcine models for medical research","volume":"40","author":"Gao","year":"2021","journal-title":"DNA Cell Biol."},{"key":"ref_2","first-page":"1","article-title":"Research progress of CRISPR-Cas system application","volume":"15","author":"Liu","year":"2020","journal-title":"Chronic Dis. Prev. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Salvesen, H.A., Grupen, C.G., and McFarlane, G.R. (2024). Tackling mosaicism in gene edited livestock. Front. Anim. Sci., 5.","DOI":"10.3389\/fanim.2024.1368155"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1038\/nrg3424","article-title":"A genomic view of mosaicism and human disease","volume":"14","author":"Biesecker","year":"2013","journal-title":"Nat. Rev. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ydbio.2016.07.017","article-title":"Electroporation of Cas9 protein\/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse","volume":"418","author":"Hashimoto","year":"2016","journal-title":"Dev. Biol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1016\/j.theriogenology.2024.01.040","article-title":"Oocyte electroporation prior to in vitro fertilization is an efficient method to generate single, double, and multiple knockout porcine embryos of interest in biomedicine and animal production","volume":"218","author":"Gadea","year":"2024","journal-title":"Theriogenology"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1530\/rep.1.00550","article-title":"The capability of reprogramming the male chromatin after fertilization is dependent on the quality of oocyte maturation","volume":"130","author":"Gioia","year":"2005","journal-title":"Reproduction"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"17472","DOI":"10.1038\/s41598-017-17805-0","article-title":"CRISPR\/Cas9 microinjection in oocytes disables pancreas development in sheep","volume":"7","author":"Vilarino","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"22309","DOI":"10.1038\/s41598-020-78264-8","article-title":"Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos","volume":"10","author":"Hennig","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1007\/s11626-019-00338-3","article-title":"Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes","volume":"55","author":"Hirata","year":"2019","journal-title":"Vitr. Cell. Dev. Biol. Anim."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1262\/jrd.2016-094","article-title":"Efficient mutagenesis by CRISPR\/Cas system during meiotic maturation of porcine oocytes","volume":"63","author":"Onuma","year":"2017","journal-title":"J. Reprod. Dev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/j.ydbio.2018.10.008","article-title":"Mosaicism in CRISPR\/Cas9-mediated genome editing","volume":"445","author":"Mehravar","year":"2019","journal-title":"Dev. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"7621","DOI":"10.1038\/srep07621","article-title":"Asymmetric parental genome engineering by Cas9 during mouse meiotic exit","volume":"4","author":"Suzuki","year":"2014","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"5532","DOI":"10.7150\/thno.43465","article-title":"A review of emerging physical transfection methods for CRISPR\/Cas9-mediated gene editing","volume":"10","author":"Fajrial","year":"2020","journal-title":"Theranostics"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Hirata, M., Wittayarat, M., Hirano, T., Nguyen, N.T., Le, Q.A., Namula, Z., Fahrudin, M., Tanihara, F., and Otoi, T. (2019). The relationship between embryonic development and the efficiency of target mutations in porcine endogenous retroviruses (PERVs) pol genes in porcine embryos. Animals, 9.","DOI":"10.3390\/ani9090593"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1080\/10495398.2019.1668801","article-title":"Generation of CD163-edited pig via electroporation of the CRISPR\/Cas9 system into porcine in vitro-fertilized zygotes","volume":"32","author":"Tanihara","year":"2021","journal-title":"Anim. Biotechnol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e13534","DOI":"10.1111\/asj.13534","article-title":"Introduction of a point mutation in the KRAS gene of in vitro fertilized porcine zygotes via electroporation of the CRISPR\/Cas9 system with single-stranded oligodeoxynucleotides","volume":"92","author":"Wittayarat","year":"2021","journal-title":"Anim. Sci. J."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"100241","DOI":"10.1016\/j.vas.2022.100241","article-title":"Effects of the timing of electroporation during in vitro maturation on triple gene editing in porcine embryos using CRISPR\/Cas9 system","volume":"16","author":"Namula","year":"2022","journal-title":"Vet. Anim. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"665","DOI":"10.1007\/s00441-011-1319-y","article-title":"Mammalian zona pellucida glycoproteins: Structure and function during fertilization","volume":"349","author":"Gupta","year":"2012","journal-title":"Cell Tissue Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"6730","DOI":"10.2741\/3185","article-title":"Structural changes of the zona pellucida during fertilization and embryo development","volume":"13","author":"Familiari","year":"2008","journal-title":"Front. Biosci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1002\/vms3.659","article-title":"Zona pellucida treatment before CRISPR\/Cas9-mediated genome editing of porcine zygotes","volume":"8","author":"Namula","year":"2022","journal-title":"Vet. Med. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"556","DOI":"10.1111\/rda.14095","article-title":"Viability and developmental potential of porcine blastocysts preserved for short term in a chemically defined medium at ambient temperature","volume":"57","author":"Lin","year":"2022","journal-title":"Reprod. Domest. Anim."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"356","DOI":"10.1262\/jrd.2024-054","article-title":"Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR\/Cas9 system","volume":"70","author":"Lin","year":"2024","journal-title":"J. Reprod. Dev."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2210","DOI":"10.14202\/vetworld.2022.2210-2216","article-title":"Multiple gene editing in porcine embryos using a combination of microinjection, electroporation, and transfection methods","volume":"15","author":"Le","year":"2022","journal-title":"Vet. World"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e1600803","DOI":"10.1126\/sciadv.1600803","article-title":"Somatic cell reprogramming-free generation of genetically modified pigs","volume":"2","author":"Tanihara","year":"2016","journal-title":"Sci. Adv."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"982","DOI":"10.1038\/nmeth.3543","article-title":"CRISPRscan: Designing highly efficient sgRNAs for CRISPR-Cas9 targeting","volume":"12","author":"Vejnar","year":"2015","journal-title":"Nat. Methods"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Uusi-M\u00e4kel\u00e4, M.I.E., Barker, H.R., B\u00e4uerlein, C.A., H\u00e4kkinen, T., Nykter, M., and R\u00e4met, M. (2018). Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio). PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0196238"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"E8","DOI":"10.1038\/s41586-018-0380-z","article-title":"Large deletions induced by Cas9 cleavage","volume":"560","author":"Adikusuma","year":"2018","journal-title":"Nature"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e70011","DOI":"10.1111\/rda.70011","article-title":"Effects of Electroporation Timing and Cumulus Cell Attachment on In Vitro Development and Genome Editing of Porcine Embryos","volume":"60","author":"Torigoe","year":"2025","journal-title":"Reprod. Domest. Anim."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"107874","DOI":"10.1016\/j.anireprosci.2025.107874","article-title":"Optimizing gene editing in pigs: The role of electroporation and lipofection","volume":"278","author":"Gadea","year":"2025","journal-title":"Anim. Reprod. Sci."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Peng, H., Wu, Y., and Zhang, Y. (2012). Efficient delivery of DNA and morpholinos into mouse preimplantation embryos by electroporation. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0043748"},{"key":"ref_32","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_33","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1002\/(SICI)1098-2795(199811)51:3<346::AID-MRD15>3.0.CO;2-0","article-title":"Functional analysis of activation of porcine oocytes by spermatozoa, calcium ionophore, and electrical pulse","volume":"51","author":"Wang","year":"1998","journal-title":"Mol. Reprod. Dev. Inc. Gamete Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1016\/j.theriogenology.2024.01.041","article-title":"Optimizing oocyte electroporation for genetic modification of porcine embryos: Evaluation of the parthenogenetic activation","volume":"218","author":"Gadea","year":"2024","journal-title":"Theriogenology"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Li, C., Zhou, S.W., Li, Y., Li, G.W., Ding, Y.G., Li, L., Liu, J., Qu, L., Sonstegard, T., and Huang, X.X. (2018). Trio-Based Deep Sequencing Reveals a Low Incidence of Off-Target Mutations in the Offspring of Genetically Edited Goats. Front. Genet., 9.","DOI":"10.3389\/fgene.2018.00449"}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/12\/1919\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,27]],"date-time":"2026-06-27T04:44:07Z","timestamp":1782535447000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/12\/1919"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,20]]},"references-count":35,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["ani16121919"],"URL":"https:\/\/doi.org\/10.3390\/ani16121919","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,20]]}}}