{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T10:08:31Z","timestamp":1792663711066,"version":"4.5.0"},"posted":{"date-parts":[[2026,7,31]]},"group-title":"Developmental Biology","reference-count":61,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T00:00:00Z","timestamp":1785456000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"id":[{"id":"https:\/\/ror.org\/03vek6s52","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/006w34k90","id-type":"ROR","asserted-by":"publisher"}]},{"award":["IOS-2220747"],"award-info":[{"award-number":["IOS-2220747"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]},{"award":["1R01GM143611,"],"award-info":[{"award-number":["1R01GM143611,"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["LT0048\/2025-L"],"award-info":[{"award-number":["LT0048\/2025-L"]}],"id":[{"id":"https:\/\/ror.org\/02ebx7v45","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,31]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Insects show extreme diversity and have long intrigued biologists. Recent technological advancements, such as gene editing and transgenesis, should in principle enable the use of almost any insect species for biological research. However, in practice, species-specific challenges remain and conditions must be optimized carefully. Here, using the milkweed bug\n                  <jats:italic>Oncopeltus fasciatus<\/jats:italic>\n                  , we first generate a useful eye\u2013 and body-color mutant strain by using CRISPR\/Cas9-mediated genome editing. Then we use this strain to develop an efficient\n                  <jats:italic>piggyBac<\/jats:italic>\n                  -mediated transgenesis system using a nuclear targeting approach. We show that incorporating 1\u00d7 and 3\u00d7 nuclear localization signals (NLS) into\n                  <jats:italic>piggyBac<\/jats:italic>\n                  mRNA substantially enhances overall transformation efficiency in\n                  <jats:italic>O. fasciatus<\/jats:italic>\n                  . Taking advantage of both the useful mutant strain and the efficient transgenesis system, we successfully integrated multiple expression cassettes ranging from 1.8 to 7.2 kb, including attP strains for phiC31-mediated site-specific integration and histone-labelled strains for live fluorescence imaging. We further provide evidence that the Q system, a binary expression system, is functional in this species, paving the way for future sophisticated genetic manipulations including functional assays for\n                  <jats:italic>cis<\/jats:italic>\n                  -regulatory elements. Together, our results not only expand the genetic toolkit of\n                  <jats:italic>O. fasciatus<\/jats:italic>\n                  as a comparative model insect, but also provide a practical framework for developing efficient transgenesis in other non-traditional model organisms.\n                <\/jats:p>","DOI":"10.64898\/2026.07.30.741915","type":"posted-content","created":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T16:40:11Z","timestamp":1785516011000},"source":"Crossref","is-referenced-by-count":1,"title":["A nuclear targeting approach enables efficient transgenesis in the milkweed bug\n                  <i>Oncopeltus fasciatus<\/i>"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-0346-7873","authenticated-orcid":false,"given":"Yu","family":"Shirai","sequence":"first","affiliation":[{"name":"Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-4486-1922","authenticated-orcid":false,"given":"Yousuf","family":"Hashmi","sequence":"additional","affiliation":[{"name":"Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA"},{"name":"Committee on Regeneration, Development, and Stem Cell Biology, University of Chicago, Chicago, IL, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ashley","family":"Watts","sequence":"additional","affiliation":[{"name":"Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1701-3265","authenticated-orcid":false,"given":"Jonchee A.","family":"Kao","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA"},{"name":"Department of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2922-5855","authenticated-orcid":false,"given":"Cassandra G.","family":"Extavour","sequence":"additional","affiliation":[{"name":"Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA"},{"name":"Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA"},{"name":"Howard Hughes Medical Institute, Chevy Chase, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026080313001600000_2026.07.30.741915v2.1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/30.11.2299"},{"key":"2026080313001600000_2026.07.30.741915v2.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ydbio.2005.04.034"},{"key":"2026080313001600000_2026.07.30.741915v2.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.ydbio.2005.08.010"},{"key":"2026080313001600000_2026.07.30.741915v2.4","doi-asserted-by":"publisher","DOI":"10.1186\/s12862-018-1293-z"},{"key":"2026080313001600000_2026.07.30.741915v2.5","doi-asserted-by":"publisher","DOI":"10.1242\/dev.096271"},{"key":"2026080313001600000_2026.07.30.741915v2.6","doi-asserted-by":"publisher","DOI":"10.1038\/46463"},{"key":"2026080313001600000_2026.07.30.741915v2.7","doi-asserted-by":"crossref","first-page":"16","DOI":"10.3390\/biom13010016","article-title":"Use of Insect Promoters in Genetic Engineering to Control Mosquito-Borne Diseases","volume":"13","year":"2022","journal-title":"Biomolecules"},{"key":"2026080313001600000_2026.07.30.741915v2.8","doi-asserted-by":"crossref","unstructured":"Chen, X. and Palli, S. 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