Generating Cisgenic Sexing Strains in Insect Pests

Generating Cisgenic Sexing Strains in Insect Pests | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Generating Cisgenic Sexing Strains in Insect Pests Angela Meccariello This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6449302/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Insect pest population control via sterile insect technique severely benefits from separation by sex prior to release. To simplify this process, traditional genetics has been deployed to develop genetic sexing strains (GSSs) for several disease vectors and agricultural pests of vast economic significance, although very few are applied in the field due to associated fitness costs and instability. In this study, we generated a method to engineer cisgenic GSS (CGSS) in insects. We use CRISPR/Cas9-mediated homology-directed repair to seamlessly translocate a sex-specific alternatively spliced intron into a dominant phenotypic gene generating a genetically stable strain that enables sex-sorting by eye. To achieve this feat, we use Ceratitis capitata as our model and relied on the sex-specifically spliced intron of the endogenous transformer gene, which we seamlessly inserted into the pupal colouration white pupae gene. This minimal modification resulted in the generation of a homozygous strain we term IMPERIAL that was phenotypically stable where all female pupae are brown while male pupae are white with overall good fitness. By minimally editing the genome, our CGSS approach can be applied to other pests that may aid more efficient and economically suitable pest control. Biological sciences/Molecular biology/CRISPR-Cas systems/CRISPR-Cas9 genome editing Biological sciences/Genetics CRISPR/Cas9 Sex Specific Alternative Splicing Genetic Sexing Strains Medfly Figures Figure 1 Figure 2 Full Text Additional Declarations Yes there is potential Competing Interest. A patent has been filed on this technology. O.S.A is a founder of Agragene, Inc. and Synvect, Inc. with equity interest. N.P.K is a founder of Synvect, Inc. with equity interest. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. All other authors declare no competing interests. Table 1 is available in the Supplementary Files section. Supplementary Files Table.pdf WPsourcedata.xlsx source data supplementarymaterials.pdf Supplementary Figures Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6449302","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":444892518,"identity":"c8f58821-f36a-44a1-817b-aa5a1d4b5fb1","order_by":0,"name":"Angela Meccariello","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9706-5764","institution":"Imperial College London","correspondingAuthor":true,"prefix":"","firstName":"Angela","middleName":"","lastName":"Meccariello","suffix":""}],"badges":[],"createdAt":"2025-04-14 22:20:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6449302/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6449302/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81021118,"identity":"38838c84-76a3-4c45-bbc4-b7e8a6a3f799","added_by":"auto","created_at":"2025-04-21 09:45:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":113047,"visible":true,"origin":"","legend":"\u003cp\u003eDesign behind the cisgenic IMPERIAL strain.\u003c/p\u003e\n\u003cp\u003e(A) A simplified diagram showcasing the IMPERIAL strain generation and its underlying mechanism. The knock-in, mediated via homology-directed repair was performed into the Benakeion wild-type strain. Due to the presence of premature stop codons in the male-specific exons, the males are phenotypically whitepupaed, whilst in females, gene rescue occurs resulting in a brown-pupae phenotype (E1-E4, Exons 1-4; LHA, left homology arm; MFS, Major Facilitator Superfamily; RHA, right homology arm; tra, transformer; wp, white pupae). (B) Graphic summary of IMPERIAL strain establishment via outcrosses to the irradiationgenerated homozygous recessive white pupae mutant (wp-/-) strain (KI, knock-in).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6449302/v1/3b02e37f0e373af99917200d.jpg"},{"id":81021117,"identity":"b2ec32f7-a24a-4944-a90f-a53d040645cc","added_by":"auto","created_at":"2025-04-21 09:45:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":117694,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterisation of the cisgenic IMPERIAL sexing strain.\u003c/p\u003e\n\u003cp\u003e(A) Stack graphs displaying pupal colour and adult phenotypes in IMPERIAL and VIENNA 8 strains for five consecutive generations (F2-F6). Chi-squared test significance levels are indicated as follows: p \u0026lt; 0.05 = *, p \u0026lt; 0.01 = **, p \u0026lt; 0.001 = ***. (B) Bar charts showing egg-adult survival of the IMPERIAL strain compared to both it parental wild-type Benakeion, and VIENNA 8 strains, completed in biological triplicates. Egg-adult survival was measured using 5-hour collections of eggs and their subsequent hatching rates, hatched larvalpupal recovery rates, and pupal-adult recovery rates. Bar levels represent mean values, whilst individual replicate values are shown with dots. Dunn’s test significance levels are indicated as follows: p \u0026lt; 0.05 = *, p \u0026lt; 0.01 = **. (C) Survival curves (Kaplan-Meier) of adult males and females from IMPERIAL, VIENNA 8 and wild-type Benakeion strains. The 95% confidence intervals are displayed using pale shading for each test group. (D) Proportion of eclosing males and females from IMPERIAL, VIENNA 8 and wild-type Benakeion strains measured daily from age-matched triplicate 24-hour egg collections. Dots represent mean values of the replicates and standard error is indicated using whiskers. (A-D) were constructed in RStudio. Source data is included in the source data file.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6449302/v1/3ba69e50126b07d2eee5cfd8.jpg"},{"id":90842323,"identity":"22ca4c07-a85b-4032-9943-bbb57bffe10e","added_by":"auto","created_at":"2025-09-08 20:50:25","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":546017,"visible":true,"origin":"","legend":"Article File","description":"","filename":"MANUSCRIPT.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6449302/v1_covered_833178b5-de5d-4fcc-9c80-b259ceef3a51.pdf"},{"id":81021116,"identity":"00259e28-e20e-4f8d-a661-8330069e9554","added_by":"auto","created_at":"2025-04-21 09:45:05","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23610,"visible":true,"origin":"","legend":"","description":"","filename":"Table.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6449302/v1/d9994230bccd71a7e3c78780.pdf"},{"id":81021119,"identity":"cf26c325-4bec-4496-be54-3620cc04e6f5","added_by":"auto","created_at":"2025-04-21 09:45:05","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20013,"visible":true,"origin":"","legend":"source data","description":"","filename":"WPsourcedata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6449302/v1/f1c53f51acf5f4d620a94dda.xlsx"},{"id":81021123,"identity":"e3d9c9d6-a116-4c56-9f9e-62e078f16c10","added_by":"auto","created_at":"2025-04-21 09:45:05","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2612422,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"supplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6449302/v1/cde75d62f10c8d6028c6b201.pdf"}],"financialInterests":"\u003cp\u003e\u003cstrong\u003eYes\u003c/strong\u003e there is potential Competing Interest. A patent has been filed on this technology. O.S.A is a founder of Agragene, Inc. and Synvect, Inc. with equity interest. N.P.K is a founder of Synvect, Inc. with equity interest. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. All other authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e","formattedTitle":"\u003cp\u003eGenerating Cisgenic Sexing Strains in Insect Pests\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CRISPR/Cas9, Sex Specific Alternative Splicing, Genetic Sexing Strains, Medfly","lastPublishedDoi":"10.21203/rs.3.rs-6449302/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6449302/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Insect pest population control via sterile insect technique severely benefits from separation by sex prior to release. To simplify this process, traditional genetics has been deployed to develop genetic sexing strains (GSSs) for several disease vectors and agricultural pests of vast economic significance, although very few are applied in the field due to associated fitness costs and instability. In this study, we generated a method to engineer cisgenic GSS (CGSS) in insects. We use CRISPR/Cas9-mediated homology-directed repair to seamlessly translocate a sex-specific alternatively spliced intron into a dominant phenotypic gene generating a genetically stable strain that enables sex-sorting by eye. To achieve this feat, we use Ceratitis capitata as our model and relied on the sex-specifically spliced intron of the endogenous transformer gene, which we seamlessly inserted into the pupal colouration white pupae gene. This minimal modification resulted in the generation of a homozygous strain we term IMPERIAL that was phenotypically stable where all female pupae are brown while male pupae are white with overall good fitness. By minimally editing the genome, our CGSS approach can be applied to other pests that may aid more efficient and economically suitable pest control.","manuscriptTitle":"Generating Cisgenic Sexing Strains in Insect Pests","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 09:45:00","doi":"10.21203/rs.3.rs-6449302/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ec6500fc-1e2b-4740-9554-fc04682a00b3","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47440733,"name":"Biological sciences/Molecular biology/CRISPR-Cas systems/CRISPR-Cas9 genome editing"},{"id":47440734,"name":"Biological sciences/Genetics"}],"tags":[],"updatedAt":"2025-09-08T20:42:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-21 09:45:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6449302","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6449302","identity":"rs-6449302","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}