TRU-PE: A Universal, Trackable Prime Editor Toolkit for Robust Single- and Multi-Locus Genome Engineering

TRU-PE: A Universal, Trackable Prime Editor Toolkit for Robust Single- and Multi-Locus Genome Engineering | 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 TRU-PE: A Universal, Trackable Prime Editor Toolkit for Robust Single- and Multi-Locus Genome Engineering Zhuobin Liang, Zhichao Qiu, Keke Sun, Qingwei Zeng, Ziwei Luo, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7705396/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 Prime editing (PE) holds immense promise for precise genome editing, but its wider adoption is constrained by large payloads and inefficient delivery in recalcitrant cell types. Here, we present TRU-PE (Trackable, Robust, and Universal Prime Editor), a toolkit that overcomes these barriers via a split-vector architecture coupled with fluorescence-guided enrichment. This design ensures stoichiometric component expression, yielding marked improvements in editing efficiency and accelerated workflows compatible with both plasmid and viral delivery. By integrating a polycistronic hCtRNA-sgRNA/pegRNA array, the derivative TRU-MPE system enables simultaneous editing of up to ten genomic loci. Furthermore, it enhances the performance of evolved PE editors, allows flexible vector stoichiometry and inspires novel editing reporters. Leveraging these capabilities, we successfully generated a combinatorial human iPSC-based platform for GATA2 deficiency, which accurately recapitulates genotype-specific hematopoietic defects. TRU-PE thus provides a user-friendly, scalable framework for robust single- and multi-locus genome engineering, accelerating functional genomics and therapeutic development. Biological sciences/Biological techniques/Genetic engineering Biological sciences/Genetics Health sciences/Medical research/Experimental models of disease Full Text Additional Declarations Yes there is potential Competing Interest. Z.L, Q.Z. and S.K. are inventors on a patent application (202411625725X) filed by Shenzhen Bay Laboratory that covers the design, composition and application of the split prime editing system and the fluorescence-guided enrichment strategy relating to this work. Other authors have no potential competing interests to declare. Supplementary Files SupplementaryTable1Plasmidsinformation.xlsx Supplementary Table 1 SupplementaryTable2Primers.xlsx Supplementary Table 2 SupplementaryTable3NGSAmpliconseq.xlsx Supplementary Table 3 SupplementaryTable4NGSpool.xlsx Supplementary Table 4 SupplementaryTable5NGSSinglecell.xlsx Supplementary Table 5 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-7705396","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":615501564,"identity":"3302b7a7-4abb-44fb-896e-f6d7352c9b6d","order_by":0,"name":"Zhuobin Liang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYDACCQYGZgYDBgY+ZhCvAojZGRgYG4jRwgbWcoaBgYeZKC1AwAYiGNuI0CI/u8fwcUHBHQY2duZnD7/Os0ncz8x88OEMBjs5XRz6GOecMTaeYfAM6DA2c2PZbWmJPcxsyYYbGJKNzQ5g18IskWMmzWNwGOQXM2nJbYeBWnjMJB8wHEjchkMLG0IL+zdpyTlEaOFBaAGq/NgA1bIBjxYJibRioF8O8wC1lEkzHEsz7jkM9MsMA9x+kZ+RvPFxwZ/Dcvz8x7dJ/qixkW1vbz74sKfCTg6XFrgDwWHBA+cb4FcOB4w/iFQ4CkbBKBgFIwsAADs8TIGquylEAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5121-2113","institution":"Shenzhen Bay Laboratory","correspondingAuthor":true,"prefix":"","firstName":"Zhuobin","middleName":"","lastName":"Liang","suffix":""},{"id":615501565,"identity":"6581b9a0-ad2c-4838-8a3e-f6829a06571b","order_by":1,"name":"Zhichao Qiu","email":"","orcid":"","institution":"IDIBELL","correspondingAuthor":false,"prefix":"","firstName":"Zhichao","middleName":"","lastName":"Qiu","suffix":""},{"id":615501566,"identity":"845d1664-c283-46e7-9d68-d3a735a66e44","order_by":2,"name":"Keke Sun","email":"","orcid":"","institution":"Institute of Molecular Physiology, Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Keke","middleName":"","lastName":"Sun","suffix":""},{"id":615501567,"identity":"839e2fd5-d1b9-4d49-aa2c-d19582e2d782","order_by":3,"name":"Qingwei Zeng","email":"","orcid":"","institution":"Institute of Molecular Physiology, Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Qingwei","middleName":"","lastName":"Zeng","suffix":""},{"id":615501568,"identity":"e225802f-d1ad-435f-a0b8-862aeebd5c8d","order_by":4,"name":"Ziwei Luo","email":"","orcid":"","institution":"Institute of Molecular Physiology, Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Ziwei","middleName":"","lastName":"Luo","suffix":""},{"id":615501569,"identity":"38354ce3-e93f-4af9-90c4-9768914bec37","order_by":5,"name":"Xinran Liu","email":"","orcid":"","institution":"Institute of Molecular Physiology, Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Xinran","middleName":"","lastName":"Liu","suffix":""},{"id":615501570,"identity":"8de52d33-e879-40f2-9666-fad07bce5908","order_by":6,"name":"Yaping Li","email":"","orcid":"","institution":"Institute of Molecular Physiology, Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Li","suffix":""},{"id":615501571,"identity":"0cb6729d-4e6b-4f38-becc-f1b8c0956a34","order_by":7,"name":"Xiang Lei","email":"","orcid":"","institution":"Institute of Molecular Physiology, Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Lei","suffix":""},{"id":615501572,"identity":"e0557694-b786-43e3-ad9f-82f5da97a20d","order_by":8,"name":"Ruilin Zhao","email":"","orcid":"","institution":"Institute of Molecular Physiology, Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Ruilin","middleName":"","lastName":"Zhao","suffix":""},{"id":615501573,"identity":"047101c1-1857-4590-8b11-c64a9443d670","order_by":9,"name":"Zhen Zhang","email":"","orcid":"","institution":"Institute of Molecular Physiology, Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Zhang","suffix":""},{"id":615501574,"identity":"a2f97fbb-2873-4895-9d87-def2c497f8a4","order_by":10,"name":"Xuan Wang","email":"","orcid":"","institution":"Institute of Molecular Physiology, Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Wang","suffix":""},{"id":615501575,"identity":"85ad4f9e-3d98-439a-a0d0-a631e0eac20c","order_by":11,"name":"Ehsan Hashemi","email":"","orcid":"","institution":"Institute of Molecular Physiology, Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Ehsan","middleName":"","lastName":"Hashemi","suffix":""},{"id":615501576,"identity":"5b183b7f-b9a6-4a22-8484-88bdab28eaeb","order_by":12,"name":"Damià Romero-Moya","email":"","orcid":"https://orcid.org/0000-0001-9947-3577","institution":"Bellvitge Institute for Biomedical Research","correspondingAuthor":false,"prefix":"","firstName":"Damià","middleName":"","lastName":"Romero-Moya","suffix":""},{"id":615501577,"identity":"75f5e213-ccbc-46dc-a195-1333b9f59844","order_by":13,"name":"Joe Z. Zhang","email":"","orcid":"https://orcid.org/0000-0002-6615-290X","institution":"Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Joe","middleName":"Z.","lastName":"Zhang","suffix":""},{"id":615501578,"identity":"ee5ec583-4906-4be8-8f83-b0042e7d9ca8","order_by":14,"name":"Xiaolong Wang","email":"","orcid":"https://orcid.org/0000-0003-1620-1344","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Xiaolong","middleName":"","lastName":"Wang","suffix":""},{"id":615501579,"identity":"33f42da3-a431-4e7f-a337-472cfe5ae264","order_by":15,"name":"Alessandra Giorgetti","email":"","orcid":"https://orcid.org/0000-0001-5803-3567","institution":"IDIBELL","correspondingAuthor":false,"prefix":"","firstName":"Alessandra","middleName":"","lastName":"Giorgetti","suffix":""},{"id":615501580,"identity":"74816c3e-b42b-4912-a284-450ff91c9105","order_by":16,"name":"Yuan Yang","email":"","orcid":"","institution":"Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-09-24 15:50:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7705396/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7705396/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106208487,"identity":"b7eb4e8c-0723-4974-853b-d96f282e6e98","added_by":"auto","created_at":"2026-04-06 06:25:55","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3119363,"visible":true,"origin":"","legend":"Article File","description":"","filename":"ArticleFileUpdated.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7705396/v1_covered_d16a981d-a364-473d-9f09-8e074eb28b26.pdf"},{"id":106208478,"identity":"29fff9c4-853e-4965-9adb-efcaf6c69975","added_by":"auto","created_at":"2026-04-06 06:25:48","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19311,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 1\u003c/p\u003e","description":"","filename":"SupplementaryTable1Plasmidsinformation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7705396/v1/560b3a645dfa1b5d5edfc56e.xlsx"},{"id":106208479,"identity":"56ee57fd-8be9-457e-af6b-4b5378d9f8b1","added_by":"auto","created_at":"2026-04-06 06:25:48","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17844,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 2\u003c/p\u003e","description":"","filename":"SupplementaryTable2Primers.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7705396/v1/c03958c32690748ee4100f10.xlsx"},{"id":106208477,"identity":"4a00d137-34e1-45aa-81b5-6165564e2e86","added_by":"auto","created_at":"2026-04-06 06:25:48","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12315,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 3\u003c/p\u003e","description":"","filename":"SupplementaryTable3NGSAmpliconseq.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7705396/v1/7cddadacd6f5adaed109e1d2.xlsx"},{"id":106208480,"identity":"f16242d7-ccbd-458f-99b8-456acb5f0155","added_by":"auto","created_at":"2026-04-06 06:25:48","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":15372,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 4\u003c/p\u003e","description":"","filename":"SupplementaryTable4NGSpool.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7705396/v1/628e4a75ba86d52622de1a88.xlsx"},{"id":106208476,"identity":"a7555b8b-b6a0-4cff-bacb-2dd542776004","added_by":"auto","created_at":"2026-04-06 06:25:47","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":11599,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 5\u003c/p\u003e","description":"","filename":"SupplementaryTable5NGSSinglecell.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7705396/v1/b16717441e0584c6a87e7896.xlsx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nZ.L, Q.Z. and S.K. are inventors on a patent application (202411625725X) filed by Shenzhen Bay Laboratory that covers the design, composition and application of the split prime editing system and the fluorescence-guided enrichment strategy relating to this work. Other authors have no potential competing interests to declare.","formattedTitle":"TRU-PE: A Universal, Trackable Prime Editor Toolkit for Robust Single- and Multi-Locus Genome Engineering","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":"","lastPublishedDoi":"10.21203/rs.3.rs-7705396/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7705396/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Prime editing (PE) holds immense promise for precise genome editing, but its wider adoption is constrained by large payloads and inefficient delivery in recalcitrant cell types. Here, we present TRU-PE (Trackable, Robust, and Universal Prime Editor), a toolkit that overcomes these barriers via a split-vector architecture coupled with fluorescence-guided enrichment. This design ensures stoichiometric component expression, yielding marked improvements in editing efficiency and accelerated workflows compatible with both plasmid and viral delivery. By integrating a polycistronic hCtRNA-sgRNA/pegRNA array, the derivative TRU-MPE system enables simultaneous editing of up to ten genomic loci. Furthermore, it enhances the performance of evolved PE editors, allows flexible vector stoichiometry and inspires novel editing reporters. Leveraging these capabilities, we successfully generated a combinatorial human iPSC-based platform for GATA2 deficiency, which accurately recapitulates genotype-specific hematopoietic defects. TRU-PE thus provides a user-friendly, scalable framework for robust single- and multi-locus genome engineering, accelerating functional genomics and therapeutic development.","manuscriptTitle":"TRU-PE: A Universal, Trackable Prime Editor Toolkit for Robust Single- and Multi-Locus Genome Engineering","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-06 06:25:18","doi":"10.21203/rs.3.rs-7705396/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-methods","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nmeth","sideBox":"Learn more about [Nature Methods](http://www.nature.com/nmeth)","snPcode":"","submissionUrl":"","title":"Nature Methods","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f6e421b4-f244-4041-923b-cda3176bee54","owner":[],"postedDate":"April 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":65493735,"name":"Biological sciences/Biological techniques/Genetic engineering"},{"id":65493736,"name":"Biological sciences/Genetics"},{"id":65493737,"name":"Health sciences/Medical research/Experimental models of disease"}],"tags":[],"updatedAt":"2026-04-06T06:25:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-06 06:25:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7705396","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7705396","identity":"rs-7705396","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}