Epigenetic editing of marine medaka(Oryzias melastigma)fgf2 using CRISPR/dCas9-Tet1CDOA
CRISPR/d Cas9-mediated epigenetic editing offers a versatile approach for transcriptional regulation without introducing DNA strand breaks.Although this strategy has been explored in a limited number of species,its application in aquatic vertebrates remains largely uncharacterized.In this study,ten-eleven translocation methylcytosine dioxygenase 1(tet1)was cloned and molecularly characterized in marine medaka(Oryzias melastigma).Decitabine treatment identified fibroblast growth factor 2(fgf2)as a methylation-sensitive gene,with a regulatory Cp G island located within its promoter region.Subsequently,a CRISPR/d Cas9-Tet1CD activation system was constructed by fusing the catalytic domain of Tet1(Tet1CD,Ala1352–Thr2034)to d Cas9,enabling locusspecific DNA demethylation.Targeting fgf2,this CRISPR/d Cas9-Tet1CD system induced efficient and selective demethylation of the Cp G island,resulting in a maximal 2.41-fold increase in fgf2 transcript levels.Wholegenome bisulfite sequencing and transcriptomic analysis confirmed high on-target precision with minimal off-target effects.Epigenetic activation of fgf2 further modulated downstream gene networks associated with growth,promoting durable transcriptional enhancement and increased cellular proliferation.Collectively,these results establish a robust and highly specific epigenetic editing platform in marine medaka,providing a powerful tool for functional genomics studies and regulatory element analysis in aquatic models.
Lei Lin;Jing-Jing Zhang;Bing-Hua Liu;Sheng Du;Yang-Qing Zhang;Yu Yang;Chen Li;Cai-Chao Dong;Yang-Bin He;Qian Wang;Hong-Yan Wang;Chang-Wei Shao
State Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,Shandong 266071,China School of Graduate,Chinese Academy of Agricultural Sciences,Beijing 100081,ChinaState Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,Shandong 266071,ChinaState Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,Shandong 266071,ChinaState Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,Shandong 266071,ChinaState Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,Shandong 266071,ChinaState Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,Shandong 266071,ChinaState Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,Shandong 266071,ChinaState Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,Shandong 266071,ChinaState Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,Shandong 266071,ChinaState Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,Shandong 266071,China Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao Marine Science and Technology Center,Qingdao,Shandong 266237,China Hebei Key Laboratory of the Bohai Sea Fish Germplasm Resources Conservation and Utilization,Beidaihe Central Experiment Station,Chinese Academy of Fishery Sciences,Qinhuangdao,Hebei 066100,ChinaState Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,Shandong 266071,China Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao Marine Science and Technology Center,Qingdao,Shandong 266237,China Hebei Key Laboratory of the Bohai Sea Fish Germplasm Resources Conservation and Utilization,Beidaihe Central Experiment Station,Chinese Academy of Fishery Sciences,Qinhuangdao,Hebei 066100,ChinaState Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,Shandong 266071,China Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao Marine Science and Technology Center,Qingdao,Shandong 266237,China Hebei Key Laboratory of the Bohai Sea Fish Germplasm Resources Conservation and Utilization,Beidaihe Central Experiment Station,Chinese Academy of Fishery Sciences,Qinhuangdao,Hebei 066100,China
生物科学
Epigenetic editingCRISPR/dCas9Tet1DNA demethylationfgf2Oryzias melastigma
《Zoological Research》 2026 (1)
P.263-278,16
supported by the National Key R&D Program of China(2022YFD2401500,2022YFD2400100)Key Research and Development Project of Shandong Province(2024LZGC005)Taishan Scholars Program(tstp20221149)AoShan Talents Cultivation Program Supported by Qingdao National Laboratory for Marine Science and Technology(2017ASTCP-ES06)National Ten-Thousands Talents Special Support Program to C.W.S.Central Public-interest Scientific Institution Basal Research Fund,CAFS(2024XT0103,2023TD19)Key R&D Program of Hebei Province,China(21326307D)。
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