{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T01:39:56Z","timestamp":1785461996325,"version":"3.56.0"},"reference-count":50,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2026,7,15]],"date-time":"2026-07-15T00:00:00Z","timestamp":1784073600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"The National Key R&D Program of China","award":["2022YFD2400101"],"award-info":[{"award-number":["2022YFD2400101"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Genome editing by the CRISPR\/Cas9 system is widely used for production of gene-modified animals, including fish. However, efficient gene editing in fish cultured cells, in particular germline stem cells (GSCs), is challenging, likely due to the difficulty in transfecting these cells. The ricefield eel (Monopterus albus), a sequential hermaphroditic species, is a freshwater fish of significant economic value in China. In this work, we report a simple method that can achieve high gene editing efficiency in primary GSCs of fish species, including ricefield eel. High transfection efficiency (50\u201395%) is achieved in fish GSCs by using a microchannel-based cell transfection system. Gene editing efficiency of up to 60% in primary ricefield eel GSCs is achieved using an integrated CRISPR\/Cas9 vector strategy. High gene editing efficiency is also achieved in gibel carp (Carassius gibelio) GSCs and the medaka spermatogonial stem cell line SG3, demonstrating the general applicability of our method. Our data suggest that efficient transfection is key to high gene editing efficiency in fish cultured GSCs. This study establishes an efficient and reliable gene editing system for fish GSCs, which may facilitate the creation of new germplasm of genetically difficult-to-breed fish by combining GSC transplantation with gene editing techniques.<\/jats:p>","DOI":"10.3390\/ijms27146294","type":"journal-article","created":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T06:49:36Z","timestamp":1784184576000},"page":"6294","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Efficient Gene Editing in Fish Primary Germline Stem Cells"],"prefix":"10.3390","volume":"27","author":[{"given":"Yuanyuan","family":"Zhan","sequence":"first","affiliation":[{"name":"Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tingting","family":"Luo","sequence":"additional","affiliation":[{"name":"Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7902-9022","authenticated-orcid":false,"given":"Yuhua","family":"Sun","sequence":"additional","affiliation":[{"name":"Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China"},{"name":"College of Life Sciences, Huzhou Normal University, Huzhou 313000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"bio035170","DOI":"10.1242\/bio.035170","article-title":"Efficient genome editing using CRISPR\/Cas9 ribonucleoprotein approach in cultured medaka fish cells","volume":"7","author":"Liu","year":"2018","journal-title":"Biol. Open"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"672","DOI":"10.1016\/j.tig.2019.06.006","article-title":"Potential of genome editing to improve aquaculture breeding and production","volume":"35","author":"Gratacap","year":"2019","journal-title":"Trends Genet."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Gratacap, R.L., Regan, T., Dehler, C.E., Martin, S.A.M., Boudinot, P., Collet, B., and Houston, R.D. (2020). Efficient CRISPR\/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system. BMC Biotechnol., 20.","DOI":"10.1186\/s12896-020-00626-x"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1007\/s10126-016-9687-7","article-title":"Editing of the Luteinizing Hormone Gene to Sterilize Channel Catfish, Ictalurus punctatus, Using a Modified Zinc Finger Nuclease Technology with Electroporation","volume":"18","author":"Qin","year":"2016","journal-title":"Mar. Biotechnol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1007\/978-1-4939-8799-3_15","article-title":"Gene Editing in Channel Catfish via Double Electroporation of Zinc-Finger Nucleases","volume":"1867","author":"Dunham","year":"2018","journal-title":"Methods Mol. Biol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"dmm034561","DOI":"10.1242\/dmm.034561","article-title":"Cancer modeling by Transgene Electroporation in Adult Zebrafish (TEAZ)","volume":"11","author":"Callahan","year":"2018","journal-title":"Dis. Model. Mech."},{"key":"ref_7","unstructured":"Wang, Y.Z. (2020). Establishment of Five Tissue Cell Lines from Chinese Sturgeon and Identification of Germ Cell Marker Gene Dazl in Black Carp. [Ph.D. Thesis, Huazhong Agricultural University]."},{"key":"ref_8","unstructured":"Wang, L. (2021). Preliminary Study on Construction and Application of Gene Editing Technology in Japanese Flounder (Paralichthys olivaceus). [Ph.D. Thesis, University of Chinese Academy of Sciences (Institute of Oceanology)]."},{"key":"ref_9","unstructured":"Xue, T. (2017). Identification and Application of Vasa Gene in Black Carp. [Ph.D. Thesis, Huazhong Agricultural University]."},{"key":"ref_10","unstructured":"He, G.M. (2021). Preliminary Study on Transfection of Fish Caudal Fin Cells and Mature Gametes by Electroporation. [Master\u2019s Thesis, Hunan Normal University]."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"16218","DOI":"10.3390\/ijms232416218","article-title":"CRISPR\/Cas9-mediated gene editing in salmonids cells and efficient establishment of edited clonal cell lines","volume":"23","author":"Stromsnes","year":"2022","journal-title":"Int. J. Mol. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1023\/A:1010393723002","article-title":"Transfection of epithelioma papulosum cyprini (EPC) carp cells","volume":"23","author":"Lopez","year":"2001","journal-title":"Biotechnol. Lett."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Zhang, Z., Wang, J., Li, J., Liu, X., Liu, L., Zhao, C., Tao, W., Wang, D., and Wei, J. (2023). Establishment of an integrated CRISPR\/Cas9 plasmid system for simple and efficient genome editing in medaka in vitro and in vivo. Biology, 12.","DOI":"10.3390\/biology12020336"},{"key":"ref_14","first-page":"1","article-title":"Spermatogenesis","volume":"13","author":"Loveland","year":"1998","journal-title":"Hum. Reprod."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1016\/j.stem.2012.05.016","article-title":"Germline stem cells: Origin and destiny","volume":"10","author":"Lehmann","year":"2012","journal-title":"Cell Stem Cell"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"8011","DOI":"10.1073\/pnas.0308668101","article-title":"Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro","volume":"101","author":"Hong","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1111\/j.1365-2443.2012.01589.x","article-title":"Production of zebrafish offspring from cultured spermatogonial stem cells","volume":"17","author":"Kawasaki","year":"2012","journal-title":"Genes Cells"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.ygcen.2014.06.018","article-title":"Biology and identity of fish spermatogonial stem cell","volume":"207","author":"Lacerda","year":"2014","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"4426","DOI":"10.7150\/ijbs.63266","article-title":"Successful spermatogonial stem cells transplantation within pleuronectiformes: First breakthrough at inter-family level in marine fish","volume":"17","author":"Zhou","year":"2021","journal-title":"Int. J. Biol. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"537","DOI":"10.24272\/j.issn.2095-8137.2022.058","article-title":"Production of functional sperm from in vitro-cultured premeiotic spermatogonia in a marine fish","volume":"43","author":"Zhang","year":"2022","journal-title":"Zool. Res."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Sun, X., Tao, B., Wang, Y., Hu, W., and Sun, Y. (2022). Isolation and characterization of germline stem cells in protogynous hermaphroditic Monopterus albus. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23115861"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Majhi, S.K., Hattori, R.S., Rahman, S.M., and Str\u00fcssmann, C.A. (2014). Surrogate production of eggs and sperm by intrapapillary transplantation of germ cells in cytoablated adult fish. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0095294"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"736977","DOI":"10.1016\/j.aquaculture.2021.736977","article-title":"Production of donor-derived offsprings by allogeneic transplantation of oogonia in the adult Japanese flounder (Paralichthys olivaceus)","volume":"543","author":"Ren","year":"2021","journal-title":"Aquaculture"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"eaaz6830","DOI":"10.1126\/science.aaz6830","article-title":"Mammalian in vitro gametogenesis","volume":"374","author":"Saitou","year":"2021","journal-title":"Science"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Kan, Y.T., Zhong, Y., Jawad, M., Sun, X.L., Liu, Z.J., Wang, W., and Wei, Q.W. (2022). Establishment of a Coilia nasus gonadal somatic cell line capable of sperm induction in vitro. Biology, 11.","DOI":"10.3390\/biology11071049"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"107756","DOI":"10.1016\/j.biotechadv.2021.107756","article-title":"Surrogate broodstock to enhance biotechnology research and applications in aquaculture","volume":"49","author":"Jin","year":"2021","journal-title":"Biotechnol. Adv."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Zhou, H., Liu, T., Zhang, T., Sun, Z., Xu, H., Zhang, T., Yin, Y., Li, N., Yan, T., and Kuang, Y. (2025). Establishment and Characteristics of the Spermatogonial Stem Cell Line from the Yellow River Carp (Cyprinus carpio haematopterus). Biology, 14.","DOI":"10.3390\/biology14050536"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1007\/s10126-016-9708-6","article-title":"Development of an Efficient Genome Editing Method by CRISPR\/Cas9 in a Fish Cell Line","volume":"18","author":"Dehler","year":"2016","journal-title":"Mar. Biotechnol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1016\/j.fsi.2018.02.039","article-title":"Efficient resistance to grass carp reovirus infection in JAM-A knockout cells using CRISPR\/Cas9","volume":"76","author":"Ma","year":"2018","journal-title":"Fish Shellfish Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Escobar-Aguirre, S., Arancibia, D., Escorza, A., Bravo, C., Andr\u00e9s, M.E., Zamorano, P., and Martinez, V. (2019). Development of a Bicistronic Vector for the Expression of a CRISPR\/Cas9-mCherry System in Fish Cell Lines. Cells, 8.","DOI":"10.3390\/cells8010075"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"7854","DOI":"10.1038\/s41598-021-87068-3","article-title":"An efficient vector-based CRISPR\/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters","volume":"11","author":"Hamar","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1186\/s13578-021-00618-0","article-title":"A ribonucleoprotein transfection strategy for CRISPR\/Cas9-mediated gene editing and single cell cloning in rainbow trout cells","volume":"11","author":"Zoppo","year":"2021","journal-title":"Cell Biosci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/0044-8486(93)90286-8","article-title":"Hormonal induction of precocious sex reversal in the ricefield eel, Monopterus albus","volume":"118","author":"Tao","year":"1993","journal-title":"Aquaculture"},{"key":"ref_34","first-page":"138","article-title":"Research and production status and development trends of ricefield eel (Monopterus albus) aquaculture","volume":"41","author":"Zhou","year":"2025","journal-title":"Shanghai J. Agric. Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e8415","DOI":"10.7717\/peerj.8415","article-title":"Population genetics of swamp eel in the Yangtze River: Comparative analyses between mitochondrial and microsatellite data provide novel insights","volume":"8","author":"Zhou","year":"2020","journal-title":"PeerJ"},{"key":"ref_36","first-page":"339","article-title":"An analysis of nutritive composition in muscle of Monopterus albus","volume":"24","author":"Shu","year":"2000","journal-title":"J. Fish. China"},{"key":"ref_37","first-page":"RP108272","article-title":"Trpv4 mediates temperature induced sex change in ricefield eel","volume":"14","author":"Yang","year":"2025","journal-title":"eLife"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1186\/s13293-022-00484-6","article-title":"Integrated chromatin accessibility and DNA methylation analysis to reveal the critical epigenetic modification and regulatory mechanism in gonadal differentiation of the sequentially hermaphroditic fish, Monopterus albus","volume":"13","author":"Hu","year":"2022","journal-title":"Biol. Sex Differ."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"352","DOI":"10.1016\/j.ygcen.2009.03.002","article-title":"Ovarian aromatase and estrogens: A pivotal role for gonadal sex differentiation and sex change in fish","volume":"165","author":"Guiguen","year":"2010","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"3030","DOI":"10.1210\/en.2016-1865","article-title":"Targeted Disruption of Aromatase Reveals Dual Functions of cyp19a1a During Sex Differentiation in Zebrafish","volume":"158","author":"Yin","year":"2017","journal-title":"Endocrinology"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1677\/jme.1.01566","article-title":"An evolutionary and functional analysis of FoxL2 in rainbow trout gonad differentiation","volume":"33","author":"Baron","year":"2004","journal-title":"J. Mol. Endocrinol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"712","DOI":"10.1210\/me.2006-0248","article-title":"Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein\/steroidogenic factor 1","volume":"21","author":"Wang","year":"2007","journal-title":"Mol. Endocrinol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1677\/JOE-08-0303","article-title":"Two cytochrome P450 aromatase genes in the hermaphrodite ricefield eel Monopterus albus: mRNA expression during ovarian development and sex change","volume":"199","author":"Zhang","year":"2008","journal-title":"J. Endocrinol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"2881","DOI":"10.1210\/en.2012-2220","article-title":"Epigenetic modifications during sex change repress gonadotropin stimulation of cyp19a1a in a teleost ricefield eel (Monopterus albus)","volume":"154","author":"Zhang","year":"2013","journal-title":"Endocrinology"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"6884","DOI":"10.1038\/srep06884","article-title":"Molecular cloning and analysis of gonadal expression of Foxl2 in the rice-field eel Monopterus albus","volume":"4","author":"Hu","year":"2014","journal-title":"Sci. Rep."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1186\/s13578-020-00490-4","article-title":"DNA methylation modification is associated with gonadal differentiation in Monopterus albus","volume":"10","author":"Wang","year":"2020","journal-title":"Cell Biosci."},{"key":"ref_47","first-page":"771","article-title":"Isolation, purification and transplantation of germ stem cells in Monopterus albus: A preliminary study","volume":"40","author":"Zhang","year":"2025","journal-title":"J. Dalian Ocean Univ."},{"key":"ref_48","first-page":"1417","article-title":"Application of microfluidic technology in cell transfection","volume":"27","author":"Xu","year":"2011","journal-title":"Chin. J. Bioeng."},{"key":"ref_49","unstructured":"Zhang, Z.M. (2023). Establishment of an Efficient and Simple CRISPR\/Cas9 Genome Editing System in Medaka (Oryzias latipes) In Vitro and In Vivo and Its Application in the Study of Sall4 Gene. [Master\u2019s Thesis, Southwest University]."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Luo, T.T., Zeng, G.X., Xia, C.C., Hu, Z., Xue, L., and Sun, Y. (2026). Surrogate production of ricefield eel sperm by germ cell transplantation in adult zig-zag eel. Water Biol. Secur., 100602.","DOI":"10.1016\/j.watbs.2026.100602"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/14\/6294\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T06:56:25Z","timestamp":1784184985000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/14\/6294"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,15]]},"references-count":50,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["ijms27146294"],"URL":"https:\/\/doi.org\/10.3390\/ijms27146294","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,15]]}}}