Modular gene tagging in C. elegans

Modular gene tagging in C. elegans | 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 Modular gene tagging in C. elegans Erik Jorgensen, Adam Hefel, Kevin Kruse, Kaden Wall, Soren Jorgensen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8482393/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Tagging a gene endogenously can identify when the gene is expressed and where the protein is localized. CRISPR is the primary tool for generating tags of endogenous genes, but it is error-prone and requires unique reagents for each gene and tag. Recombinases can insert DNA in an error-free and modular manner. Here, we tested eight recombinases for germline function in the nematode C. elegans, and introduce PhIT, a recombinase-based method for protein tagging. First, a short 39bp PhiC31 attB landing pad is inserted into the locus by CRISPR. This strain is a resource which can be used to insert a variety of modular tags. Second, tags are inserted by the integrase PhiC31, and in tandem, extraneous backbone sequences are removed by a tyrosine recombinase. Current modular tags include seven different fluorescent proteins, FLP-regulated cell-specific expression constructs, and degron tags. Importantly, tags can be inserted by genetic crosses instead of by microinjection. Biological sciences/Genetics/Genome Biological sciences/Biological techniques/Genetic engineering C. elegans genome engineering gene tagging PhIT PhiC31 Dre B3 recombinase Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Extendeddatatables14.xlsx Extended Data Tables Cite Share Download PDF Status: Under Review 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-8482393","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":580770010,"identity":"d5bc1e53-87fd-408a-968a-3460c3e33e9a","order_by":0,"name":"Erik Jorgensen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIiWNgGAWjYBACxgYYi72H4QBDAbLwAUJaeM4A1RhABfFpQQCJHCBBjBbm9t7HLz7uqJMzl3x78MAHAxu7DQd4zB/83MEgx3cjAbvDeo6bWc48c9jYcnZewsEZBmnJQC2Gjb1nGIwlcWmZkcZmzNt2IHHD7RyDwzwGh5MNgFoaeNsYEjfg0/K3ra5+w80zCC2Nf9sY6vFoYX7M2MacYHCDB6zFDqSlGWgLUASXX46xMfa2HTbccAbilwTJw2yFs2XbJAxnnnmAVYthexvzh59tdfIGx88e/vChwsae73jzho9v22zk+Y5jt8WwgYFNAlkgccFhMC2BVTkIyAOj5gOygL18A07Fo2AUjIJRMEIBABkTa3uVge91AAAAAElFTkSuQmCC","orcid":"","institution":"University of Utah","correspondingAuthor":true,"prefix":"","firstName":"Erik","middleName":"","lastName":"Jorgensen","suffix":""},{"id":580770011,"identity":"5422d732-ddab-4e15-ad78-e4406165efae","order_by":1,"name":"Adam Hefel","email":"","orcid":"","institution":"University of Utah","correspondingAuthor":false,"prefix":"","firstName":"Adam","middleName":"","lastName":"Hefel","suffix":""},{"id":580770012,"identity":"c70eb287-a6d9-4250-ba2f-67958d7eb36d","order_by":2,"name":"Kevin Kruse","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Kruse","suffix":""},{"id":580770013,"identity":"51bbaeae-3e5b-4733-83a3-01e454ae3317","order_by":3,"name":"Kaden Wall","email":"","orcid":"","institution":"University of Utah","correspondingAuthor":false,"prefix":"","firstName":"Kaden","middleName":"","lastName":"Wall","suffix":""},{"id":580770014,"identity":"d3bb01bf-b0ae-4c76-a184-7b3791dcf639","order_by":4,"name":"Soren Jorgensen","email":"","orcid":"","institution":"University of Utah","correspondingAuthor":false,"prefix":"","firstName":"Soren","middleName":"","lastName":"Jorgensen","suffix":""},{"id":580770015,"identity":"3ff443a7-e16e-406d-ab97-5e73698afaee","order_by":5,"name":"Kam Ng","email":"","orcid":"","institution":"University of Utah","correspondingAuthor":false,"prefix":"","firstName":"Kam","middleName":"","lastName":"Ng","suffix":""},{"id":580770016,"identity":"e821db45-0eed-47db-ad1a-8c23adbf8f9e","order_by":6,"name":"Ryan Stolley","email":"","orcid":"","institution":"University of Utah","correspondingAuthor":false,"prefix":"","firstName":"Ryan","middleName":"","lastName":"Stolley","suffix":""},{"id":580770017,"identity":"a2326f44-aa4f-4840-9b1a-9ff73b6389fc","order_by":7,"name":"Matthew Rich","email":"","orcid":"","institution":"University of Utah","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Rich","suffix":""}],"badges":[],"createdAt":"2025-12-30 14:46:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8482393/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8482393/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102298072,"identity":"8e57ad46-6a14-4bf0-a634-8adb4ac5d2b9","added_by":"auto","created_at":"2026-02-10 10:30:21","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11054066,"visible":true,"origin":"","legend":"Article File","description":"","filename":"PhITmanv88complete.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8482393/v1_covered_ffe2d2f5-57a3-4ed9-8ae4-d1cc9b237037.pdf"},{"id":102276619,"identity":"31bd6483-270d-4cc2-b8b6-c4adf97a8da0","added_by":"auto","created_at":"2026-02-10 06:01:09","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":39904,"visible":true,"origin":"","legend":"Extended Data Tables","description":"","filename":"Extendeddatatables14.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8482393/v1/3ed14d2e2256d3e925f072a8.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Modular gene tagging in C. elegans","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"C. elegans genome engineering, gene tagging, PhIT, PhiC31, Dre, B3 recombinase","lastPublishedDoi":"10.21203/rs.3.rs-8482393/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8482393/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Tagging a gene endogenously can identify when the gene is expressed and where the protein is localized. CRISPR is the primary tool for generating tags of endogenous genes, but it is error-prone and requires unique reagents for each gene and tag. Recombinases can insert DNA in an error-free and modular manner. Here, we tested eight recombinases for germline function in the nematode C. elegans, and introduce PhIT, a recombinase-based method for protein tagging. First, a short 39bp PhiC31 attB landing pad is inserted into the locus by CRISPR. This strain is a resource which can be used to insert a variety of modular tags. Second, tags are inserted by the integrase PhiC31, and in tandem, extraneous backbone sequences are removed by a tyrosine recombinase. Current modular tags include seven different fluorescent proteins, FLP-regulated cell-specific expression constructs, and degron tags. Importantly, tags can be inserted by genetic crosses instead of by microinjection.","manuscriptTitle":"Modular gene tagging in C. elegans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-10 06:01:03","doi":"10.21203/rs.3.rs-8482393/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ab36f19e-b076-453b-b997-44e47140aef2","owner":[],"postedDate":"February 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61782800,"name":"Biological sciences/Genetics/Genome"},{"id":61782801,"name":"Biological sciences/Biological techniques/Genetic engineering"}],"tags":[],"updatedAt":"2026-02-10T06:01:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-10 06:01:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8482393","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8482393","identity":"rs-8482393","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}