{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T07:26:36Z","timestamp":1786951596928,"version":"build-2736575974"},"reference-count":81,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T00:00:00Z","timestamp":1786924800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T00:00:00Z","timestamp":1786924800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100012459","name":"Chongqing Graduate Student Research Innovation Project","doi-asserted-by":"publisher","award":["CYB240114"],"award-info":[{"award-number":["CYB240114"]}],"id":[{"id":"10.13039\/501100012459","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32172797"],"award-info":[{"award-number":["32172797"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Insect Science"],"abstract":"Abstract<\/jats:title>\n \n CRISPR\/Cas9 has become a key tool for functional genomics and genetic control strategies in mosquitoes, with its efficiency highly dependent on precise spatiotemporal regulation of Cas9 expression. While germline\u2010specific promoters help reduce resistance and improve drive efficiency, constitutive promoters confer higher editing efficiency due to their ubiquitous and sustained expression, making them more suitable for gene functional analysis and population\u2010specific suppression. However, the scarcity of strong constitutive promoters in\n Aedes aegypti<\/jats:italic>\n (\n Ae. aegypti<\/jats:italic>\n ) has limited the development of highly efficient editing systems. In this study, we cloned the promoter of the housekeeping gene\n eukaryotic translation elongation factor 1\u03b1<\/jats:italic>\n (\n EF1\u0391<\/jats:italic>\n , AAEL017096) and confirmed its transcriptional activity. RT\u2010qPCR analysis revealed that the\n EF1\u0391<\/jats:italic>\n promoter (\n EF1\u0391<\/jats:italic>\n p) directed significantly higher Cas9 expression in mosquito ovaries and testes than did the germline\u2010specific\n Exu<\/jats:italic>\n promoter (\n Exu<\/jats:italic>\n p). Crossing\n EF1A<\/jats:italic>\n p\u2010\n Cas9<\/jats:italic>\n transgenic lines with various gRNA\u2010expressing lines achieved efficient editing of the marker gene\n white<\/jats:italic>\n , the functional gene\n PNP<\/jats:italic>\n , and the microRNA precursor miR\u20101174, with all double\u2010positive progeny displaying expected loss\u2010of\u2010function phenotypes. To overcome vector capacity limitations, we truncated the\n U6<\/jats:italic>\n promoter. A truncated\n U6<\/jats:italic>\n promoter of only 235 bp (\n U6<\/jats:italic>\n p235) supported editing efficiency comparable to the full\u2010length 964 bp version. When\n U6<\/jats:italic>\n p235\u2010white gRNA lines were crossed with\n EF1A<\/jats:italic>\n p\u2010\n Cas9<\/jats:italic>\n lines, all double\u2010positive offspring exhibited a white\u2010eye phenotype, confirming successful white gene disruption. Together, this work provides an efficient constitutive promoter and a compact\n U6<\/jats:italic>\n p, expanding the genetic toolkit for\n Ae. aegypti<\/jats:italic>\n and supporting advanced functional studies and multi\u2010target control strategies.\n <\/jats:p>","DOI":"10.1111\/1744-7917.70338","type":"journal-article","created":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T07:08:43Z","timestamp":1786950523000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A potent\n EF1A<\/i>\n promoter and a minimal\n U6<\/i>\n promoter enable highly efficient CRISPR genome editing in\n Aedes aegypti<\/i>"],"prefix":"10.1111","author":[{"given":"Xiaolin","family":"Yang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Resource Insects Southwest University Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Houming","family":"Ren","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Resource Insects Southwest University Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meng","family":"Huang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Resource Insects Southwest University Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenzhuo","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Resource Insects Southwest University Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yujiao","family":"Han","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Resource Insects Southwest University Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuqi","family":"Lou","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Resource Insects Southwest University Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2917-0172","authenticated-orcid":false,"given":"Shiping","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Resource Insects Southwest University Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,8,17]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2583.2010.01005.x"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.9b00436"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2312456121"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2164-12-606"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2013.06.020"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/46463"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2004373117"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.aaz0525"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0007948"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2105075118"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10340-023-01736-z"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2164-7-120"},{"key":"e_1_2_9_14_1","doi-asserted-by":"crossref","first-page":"1126","DOI":"10.1038\/s42003-024-06848-5","article-title":"Expanding the CRISPR base editing toolbox in Drosophila melanogaster","volume":"7","author":"Clark M.","year":"2024","journal-title":"Communications Biology"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gky354"},{"key":"e_1_2_9_16_1","doi-asserted-by":"crossref","DOI":"10.1126\/science.ads3732","article-title":"1206 genomes reveal origin and movement of Aedes aegypti driving increased dengue risk","volume":"389","author":"Crawford J.E.","year":"2025","journal-title":"Science"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cois.2015.04.012"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00047711"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.5b00299"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1093\/oxfordjournals.molbev.a025920"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1111\/1744-7917.70038"},{"key":"e_1_2_9_22_1","doi-asserted-by":"publisher","DOI":"10.1111\/1744-7917.13371"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-48874-1"},{"key":"e_1_2_9_24_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-23239-0"},{"key":"e_1_2_9_25_1","doi-asserted-by":"crossref","DOI":"10.1073\/pnas.2004838117","article-title":"Analysis of off\u2010target effects in CRISPR\u2010based gene drives in the human malaria mosquito","volume":"118","author":"Garrood W.T.","year":"2021","journal-title":"Proceedings of the National Academy of Sciences USA"},{"key":"e_1_2_9_26_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2583.1999.00139.x"},{"key":"e_1_2_9_27_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10340-025-01895-1"},{"key":"e_1_2_9_28_1","doi-asserted-by":"crossref","first-page":"3175","DOI":"10.1093\/nar\/16.8.3175","article-title":"Two genes encode related cytoplasmic elongation factors 1 alpha (EF\u20101 alpha) in Drosophila melanogaster with continuous and stage\u2010specific expression","volume":"16","author":"Hovemann B.","year":"1988","journal-title":"Nucleic Acids Research"},{"key":"e_1_2_9_29_1","doi-asserted-by":"publisher","DOI":"10.1111\/1744-7917.70190"},{"key":"e_1_2_9_30_1","doi-asserted-by":"publisher","DOI":"10.1080\/19768354.2024.2322054"},{"key":"e_1_2_9_31_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-07964-7"},{"key":"e_1_2_9_32_1","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/15.5.356"},{"key":"e_1_2_9_33_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0965-1748(01)00120-5"},{"key":"e_1_2_9_34_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-019-0376-y"},{"key":"e_1_2_9_35_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkz365"},{"key":"e_1_2_9_36_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.xpro.2021.100432"},{"key":"e_1_2_9_37_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1711538114"},{"key":"e_1_2_9_38_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.90199.3"},{"key":"e_1_2_9_39_1","doi-asserted-by":"publisher","DOI":"10.1534\/g3.118.200883"},{"key":"e_1_2_9_40_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-25421-w"},{"key":"e_1_2_9_41_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.51701"},{"key":"e_1_2_9_42_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-025-09283-6"},{"key":"e_1_2_9_43_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1416278111"},{"key":"e_1_2_9_44_1","doi-asserted-by":"publisher","DOI":"10.1111\/1744-7917.12645"},{"key":"e_1_2_9_45_1","doi-asserted-by":"publisher","DOI":"10.1006\/meth.2001.1262"},{"key":"e_1_2_9_46_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2020.107841"},{"key":"e_1_2_9_47_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13071-023-05859-1"},{"key":"e_1_2_9_48_1","doi-asserted-by":"publisher","DOI":"10.1111\/1744-7917.12609"},{"key":"e_1_2_9_49_1","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1093\/jisesa\/ieaa074","article-title":"Delivery methods for RNAi in mosquito larvae","volume":"20","author":"Munawar K.","year":"2020","journal-title":"Journal of Insect Science"},{"key":"e_1_2_9_50_1","doi-asserted-by":"publisher","DOI":"10.1111\/1744-7917.70150"},{"key":"e_1_2_9_51_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1138878"},{"key":"e_1_2_9_52_1","doi-asserted-by":"publisher","DOI":"10.1111\/pbi.13736"},{"key":"e_1_2_9_53_1","doi-asserted-by":"publisher","DOI":"10.1021\/jf200689r"},{"key":"e_1_2_9_54_1","doi-asserted-by":"publisher","DOI":"10.1534\/genetics.118.301736"},{"key":"e_1_2_9_55_1","doi-asserted-by":"publisher","DOI":"10.3390\/biom15050644"},{"key":"e_1_2_9_56_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2025.143243"},{"key":"e_1_2_9_57_1","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1186\/s13071-023-05729-w","article-title":"Mosquito diversity (Diptera: Culicidae) and medical importance in four Cambodian forests","volume":"16","author":"Rakotonirina A.","year":"2023","journal-title":"Parasites & Vectors"},{"key":"e_1_2_9_58_1","doi-asserted-by":"crossref","DOI":"10.1073\/pnas.2411587121","article-title":"Precision\u2010guided tools for malaria control","volume":"121","author":"Rasgon J.L.","year":"2024","journal-title":"Proceedings of the National Academy of Sciences USA"},{"key":"e_1_2_9_59_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0097-8485(01)00099-7"},{"key":"e_1_2_9_60_1","doi-asserted-by":"crossref","DOI":"10.1093\/g3journal\/jkac280","article-title":"Assessing single\u2010locus CRISPR\/Cas9\u2010based gene drive variants in the mosquito Aedes aegypti via single\u2010generation crosses and modeling","volume":"12","author":"Reid W.","year":"2022","journal-title":"G3: Genes, Genomes, Genetics"},{"key":"e_1_2_9_61_1","doi-asserted-by":"publisher","DOI":"10.1111\/1744\u20107917.70155"},{"key":"e_1_2_9_62_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1318481110"},{"key":"e_1_2_9_63_1","doi-asserted-by":"crossref","first-page":"576","DOI":"10.1186\/s13071-016-1859-9","article-title":"Identification of interchangeable cross\u2010species function of elongation factor\u20101 alpha promoters in Babesia bigemina and Babesia bovis","volume":"9","author":"Silva M.G.","year":"2016","journal-title":"Parasites & Vectors"},{"key":"e_1_2_9_64_1","doi-asserted-by":"publisher","DOI":"10.7717\/peerj.16287"},{"key":"e_1_2_9_65_1","doi-asserted-by":"crossref","DOI":"10.1126\/sciadv.ade8903","article-title":"A confinable female\u2010lethal population suppression system in the malaria vector Anopheles gambiae","volume":"9","author":"Smidler A.L.","year":"2023","journal-title":"Science Advances"},{"key":"e_1_2_9_66_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-60761-854-6_5"},{"key":"e_1_2_9_67_1","doi-asserted-by":"publisher","DOI":"10.1002\/humu.23819"},{"key":"e_1_2_9_68_1","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1186\/1741-7007-12-27","article-title":"Genomic composition and evolution of Aedes aegypti chromosomes revealed by the analysis of physically mapped supercontigs","volume":"12","author":"Timoshevskiy V.A.","year":"2014","journal-title":"BMC Biology"},{"key":"e_1_2_9_69_1","doi-asserted-by":"crossref","first-page":"7145","DOI":"10.1038\/s41467-022-34739-y","article-title":"A CRISPR endonuclease gene drive reveals distinct mechanisms of inheritance bias","volume":"13","author":"Verkuijl S.A.N.","year":"2022","journal-title":"Nature Communications"},{"key":"e_1_2_9_70_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(18)43956-7"},{"key":"e_1_2_9_71_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12915-022-01232-3"},{"key":"e_1_2_9_72_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.plaphy.2013.05.038"},{"key":"e_1_2_9_73_1","doi-asserted-by":"publisher","DOI":"10.1093\/jme\/tjab128"},{"key":"e_1_2_9_74_1","first-page":"780","article-title":"Validation of reference genes for RT\u2010qPCR studies of gene expression in preharvest and postharvest longan fruits under different experimental conditions","volume":"7","author":"Wu J.","year":"2016","journal-title":"Frontiers in Plant Science"},{"key":"e_1_2_9_75_1","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.jip.2018.04.011","article-title":"Validation of housekeeping genes for quantitative mRNA expression analysis in OsHV\u20101 infected ark clam, Scapharca broughtonii","volume":"155","author":"Xin L.","year":"2018","journal-title":"Journal of Invertebrate Pathology"},{"key":"e_1_2_9_76_1","doi-asserted-by":"crossref","first-page":"1007","DOI":"10.1038\/s41467-025-56290-2","article-title":"Gene drive\u2010based population suppression in the malaria vector Anopheles stephensi","volume":"16","author":"Xu X.","year":"2025","journal-title":"Nature Communications"},{"key":"e_1_2_9_77_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2018.11.019"},{"key":"e_1_2_9_78_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10340-024-01864-0"},{"key":"e_1_2_9_79_1","doi-asserted-by":"publisher","DOI":"10.1111\/1744-7917.13306"},{"key":"e_1_2_9_80_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2020.735134"},{"key":"e_1_2_9_81_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0166074"},{"key":"e_1_2_9_82_1","doi-asserted-by":"publisher","DOI":"10.1089\/crispr.2020.0052"}],"container-title":["Insect Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/1744-7917.70338","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1111\/1744-7917.70338","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/1744-7917.70338","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T07:08:46Z","timestamp":1786950526000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1111\/1744-7917.70338"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,17]]},"references-count":81,"alternative-id":["10.1111\/1744-7917.70338"],"URL":"https:\/\/doi.org\/10.1111\/1744-7917.70338","archive":["Portico"],"relation":{},"ISSN":["1672-9609","1744-7917"],"issn-type":[{"value":"1672-9609","type":"print"},{"value":"1744-7917","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,17]]},"assertion":[{"value":"2026-02-21","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-05","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-08-17","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"1744-7917.70338"}}