{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T12:28:44Z","timestamp":1786969724994,"version":"build-2736575974"},"reference-count":27,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,16]],"date-time":"2026-08-16T00:00:00Z","timestamp":1786838400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100007934","name":"Beijing Academy of Agricultural and Forestry Sciences","doi-asserted-by":"publisher","award":["KJCX20261411"],"award-info":[{"award-number":["KJCX20261411"]}],"id":[{"id":"10.13039\/501100007934","id-type":"DOI","asserted-by":"publisher"}]},{"award":["KJCX20261411"],"award-info":[{"award-number":["KJCX20261411"]}],"id":[{"id":"https:\/\/ror.org\/04trzn023","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China (NSFC) Youth Science Foundation Project","doi-asserted-by":"publisher","award":["32403015"],"award-info":[{"award-number":["32403015"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["32403015"],"award-info":[{"award-number":["32403015"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100007934","name":"Innovation ability construction project","doi-asserted-by":"publisher","award":["KJCX20230216"],"award-info":[{"award-number":["KJCX20230216"]}],"id":[{"id":"10.13039\/501100007934","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"General Program of Beijing Natural Science Foundation","doi-asserted-by":"publisher","award":["6262009"],"award-info":[{"award-number":["6262009"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100007934","name":"Construction of International Science and Technology Cooperation Platforms of Beijing Academy of Agriculture and Forestry Sciences","doi-asserted-by":"publisher","award":["GHPT2026-16"],"award-info":[{"award-number":["GHPT2026-16"]}],"id":[{"id":"10.13039\/501100007934","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100007934","name":"Beijing Key Laboratory of Fisheries Biotechnology","doi-asserted-by":"publisher","award":["PT2025-20"],"award-info":[{"award-number":["PT2025-20"]}],"id":[{"id":"10.13039\/501100007934","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Beijing Rural Revitalization Agricultural Science and Technology Project","award":["NY2401170024"],"award-info":[{"award-number":["NY2401170024"]}]},{"name":"Beijing International Science & Technology Cooperation Base","award":["GHPT2026-06"],"award-info":[{"award-number":["GHPT2026-06"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Int. J. Mol. Sci.","IJMS"],"abstract":"<jats:p>The twin-tail phenotype of goldfish represents a striking domestication-associated remodeling of the vertebrate caudal axial system and is classically linked to disruption of Chordin\/BMP-mediated dorsal\u2013ventral patterning. Although previous knockdown studies implicated sizzled (szl) in this process, genetic evidence from targeted disruption of endogenous szl loci remains limited. Here, we used CRISPR\/Cas9 to mutate conserved coding regions shared by the duplicated goldfish paralogues szlA and szlB in single-tail embryos. Sanger sequencing and ICE analysis showed that szl-sgRNA2 and szl-sgRNA3 efficiently induced indels at both loci, whereas szl-sgRNA1 was ineffective. Across three independent biological replicates, twin-tail-like caudal bifurcation was observed in 44.63\u201348.19% of szl-sgRNA2-injected larvae, 69.47\u201379.61% of szl-sgRNA3-injected larvae, and 64.29\u201376.19% of larvae injected with the sgRNA mixture; szl-sgRNA1-injected larvae remained single-tailed. Calcein staining further revealed separation of distal caudal fin rays and partial splitting of the caudal skeletal complex in szl-edited larvae. qRT-PCR showed selective remodeling of dorsal\u2013ventral patterning genes, including reduced chdA and eve1 expression and increased bmp2 and nog1 expression. These findings provide direct functional evidence that szl regulates median caudal patterning in goldfish and suggest that szl-dependent modulation of the Chordin\/BMP network can generate twin-tail-like caudal morphology.<\/jats:p>","DOI":"10.3390\/ijms27167318","type":"journal-article","created":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T12:01:26Z","timestamp":1786968086000},"page":"7318","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["CRISPR\/Cas9-Mediated Disruption of Duplicated Sizzled Genes Induces Twin-Tail-like Caudal Bifurcation in Goldfish (Carassius auratus)"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-8875-8994","authenticated-orcid":false,"given":"Huijuan","family":"Li","sequence":"first","affiliation":[{"name":"Beijing Key Laboratory of Fishery Biotechnology, Fisheries Science Institute, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoying","family":"Zhang","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Physiology, Department of Zoology, Kiel University, 24098 Kiel, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaowen","family":"Wang","sequence":"additional","affiliation":[{"name":"Beijing Key Laboratory of Fishery Biotechnology, Fisheries Science Institute, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rong","family":"Zhang","sequence":"additional","affiliation":[{"name":"Beijing Key Laboratory of Fishery Biotechnology, Fisheries Science Institute, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8608-6471","authenticated-orcid":false,"given":"Lili","family":"Liu","sequence":"additional","affiliation":[{"name":"Beijing Key Laboratory of Fishery Biotechnology, Fisheries Science Institute, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lixin","family":"Sun","sequence":"additional","affiliation":[{"name":"Fisheries College, Jimei University, Xiamen 361021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhigang","family":"Yao","sequence":"additional","affiliation":[{"name":"Beijing Key Laboratory of Fishery Biotechnology, Fisheries Science Institute, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hua","family":"Zhu","sequence":"additional","affiliation":[{"name":"Beijing Key Laboratory of Fishery Biotechnology, Fisheries Science Institute, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Smartt, J. (2001). Goldfish Varieties and Genetics: A Handbook for Breeders, Blackwell Science.","DOI":"10.1002\/9780470999790"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1002\/wdev.224","article-title":"Goldfish morphology as a model for evolutionary developmental biology","volume":"5","author":"Ota","year":"2016","journal-title":"Wiley Interdiscip. Rev. Dev. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1002\/dvdy.15","article-title":"Embryonic and postembryonic development of the ornamental twin-tail goldfish","volume":"248","author":"Li","year":"2019","journal-title":"Dev. Dyn."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/j.ydbio.2016.11.022","article-title":"Evolutionary developmental transition from median to paired morphology of vertebrate fins: Perspectives from twin-tail goldfish","volume":"427","author":"Abe","year":"2017","journal-title":"Dev. Biol."},{"key":"ref_5","first-page":"287","article-title":"A history of the domestication and the factors of the varietal formation of the common goldfish, Carassius auratus","volume":"5","author":"Chen","year":"1956","journal-title":"Sci. Sin."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3360","DOI":"10.1038\/ncomms4360","article-title":"The origin of the bifurcated axial skeletal system in the twin-tail goldfish","volume":"5","author":"Abe","year":"2014","journal-title":"Nat. Commun."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"372","DOI":"10.1002\/jez.b.22831","article-title":"A novel allele of the goldfish chdB gene: Functional evaluation and evolutionary considerations","volume":"330","author":"Abe","year":"2018","journal-title":"J. Exp. Zool. Part B Mol. Dev. Evol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/S0378-1119(01)00827-7","article-title":"Chordin-like CR domains and the regulation of evolutionarily conserved extracellular signaling systems","volume":"287","author":"Coffinier","year":"2002","journal-title":"Gene"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"442","DOI":"10.1038\/70557","article-title":"Patterning the zebrafish axial skeleton requires early chordin function","volume":"23","author":"Fisher","year":"1999","journal-title":"Nat. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"862","DOI":"10.1038\/43092","article-title":"The zebrafish organizer requires chordino","volume":"387","author":"Lee","year":"1997","journal-title":"Nature"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2298","DOI":"10.1002\/dvdy.21245","article-title":"Phenotypic analysis of a novel chordin mutant in medaka","volume":"236","author":"Takashima","year":"2007","journal-title":"Dev. Dyn."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1146\/annurev-genet-110410-132517","article-title":"Maternal and zygotic control of zebrafish dorsoventral axial patterning","volume":"45","author":"Langdon","year":"2011","journal-title":"Annu. Rev. Genet."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1002\/jez.b.22811","article-title":"An alternative evolutionary pathway for the twin-tail goldfish via szl gene mutation","volume":"330","author":"Abe","year":"2018","journal-title":"J. Exp. Zool. Part B Mol. Dev. Evol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1242\/dev.123.1.95","article-title":"dino and mercedes, two genes regulating dorsal development in the zebrafish embryo","volume":"123","author":"Hammerschmidt","year":"1996","journal-title":"Development"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1242\/dev.123.1.255","article-title":"Genetic analysis of fin formation in the zebrafish, Danio rerio","volume":"123","author":"Granato","year":"1996","journal-title":"Development"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"33581","DOI":"10.1074\/jbc.M112.380816","article-title":"Sizzled is unique among secreted Frizzled-related proteins for its ability to specifically inhibit bone morphogenetic protein-1 (BMP-1)\/Tolloid-like proteinases","volume":"287","author":"Bijakowski","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2705","DOI":"10.1242\/dev.00506","article-title":"Ogon\/Secreted Frizzled functions as a negative feedback regulator of BMP signaling","volume":"130","author":"Yabe","year":"2003","journal-title":"Development"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"699","DOI":"10.1016\/j.molcel.2018.11.031","article-title":"Target-specific precision of CRISPR-mediated genome editing","volume":"73","author":"Chakrabarti","year":"2019","journal-title":"Mol. Cell"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3422","DOI":"10.1038\/s41467-022-30480-8","article-title":"Cas9-induced large deletions and small indels are controlled in a convergent fashion","volume":"13","author":"Kosicki","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1485","DOI":"10.1002\/dvdy.24340","article-title":"Postembryonic staging of wild-type goldfish, with brief reference to skeletal systems","volume":"244","author":"Li","year":"2015","journal-title":"Dev. Dyn."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e25861","DOI":"10.7554\/eLife.25861","article-title":"Dynamics of BMP signaling and distribution during zebrafish dorsal\u2013ventral patterning","volume":"6","author":"Pomreinke","year":"2017","journal-title":"eLife"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1016\/j.cell.2005.12.018","article-title":"Embryonic dorsal\u2013ventral signaling: Secreted Frizzled-related proteins as inhibitors of Tolloid proteinases","volume":"124","author":"Lee","year":"2006","journal-title":"Cell"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"26838","DOI":"10.1038\/srep26838","article-title":"Open and closed evolutionary paths for drastic morphological changes, involving serial gene duplication, sub-functionalization, and selection","volume":"6","author":"Abe","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"19961","DOI":"10.1038\/s41598-022-24444-7","article-title":"Pleiotropic functions of chordin gene causing drastic morphological changes in ornamental goldfish","volume":"12","author":"Chen","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1006\/dbio.1999.9401","article-title":"Three different noggin genes antagonize the activity of bone morphogenetic proteins in the zebrafish embryo","volume":"214","author":"Thisse","year":"1999","journal-title":"Dev. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3865","DOI":"10.1007\/s10750-020-04414-8","article-title":"A detailed procedure for CRISPR\/Cas9-mediated gene editing in tilapia","volume":"848","author":"Li","year":"2021","journal-title":"Hydrobiologia"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e168","DOI":"10.1093\/nar\/gku936","article-title":"Easy quantitative assessment of genome editing by sequence trace decomposition","volume":"42","author":"Brinkman","year":"2014","journal-title":"Nucleic Acids Res."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/16\/7318\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T12:09:24Z","timestamp":1786968564000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/16\/7318"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,16]]},"references-count":27,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["ijms27167318"],"URL":"https:\/\/doi.org\/10.3390\/ijms27167318","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,16]]}}}