Validation of a CD81-based flow cytometry assay to assess dCas9 silencing activity

Validation of a CD81-based flow cytometry assay to assess dCas9 silencing activity | 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 Short Report Validation of a CD81-based flow cytometry assay to assess dCas9 silencing activity Manosalva, Charles-Alfred, Torres, Spicuglia This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8661827/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 The efficiency of CRISPR interference (CRISPRi) depends on functional dCas9 activity, yet practical and reproducible validation of dCas9-expressing cell lines remains limited. Here, we describe a simple and reproducible assay to assess dCas9 functionality using a single sgRNA targeting the ubiquitously expressed surface protein CD81. We evaluated this approach in multiple hematological and solid tumor cell lines expressing the dCas9-KRAB-MeCP2 repressor complex. In all tested models, CD81 targeting resulted in a consistent reduction of surface protein levels, quantified by flow cytometry. This assay provides a rapid and quantitative functional readout of dCas9 activity without the need for reporter constructs or transcriptional assays. The CD81-targeting sgRNA and validated cell lines are made available to support reproducibility and technical standardization in CRISPRi experiments. This strategy can be readily implemented in any laboratory using CRISPRi-based approaches. Full Text Additional Declarations The authors declare no competing interests. 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-8661827","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":578254307,"identity":"3ad7e05f-5c06-4408-b138-cb86a6896af3","order_by":0,"name":"Manosalva","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"","middleName":"","lastName":"Manosalva","suffix":""},{"id":578254308,"identity":"0912b15e-6379-4f5f-9cea-364d98991081","order_by":1,"name":"Charles-Alfred","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"","middleName":"","lastName":"Charles-Alfred","suffix":""},{"id":578254309,"identity":"161ba13d-f230-4c1c-b525-f03b6d1e6b77","order_by":2,"name":"Torres","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"","middleName":"","lastName":"Torres","suffix":""},{"id":578254310,"identity":"d4d66058-1e3d-413c-8bfd-bba51b5162d4","order_by":3,"name":"Spicuglia","email":"data:image/png;base64,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","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"","middleName":"","lastName":"Spicuglia","suffix":""}],"badges":[],"createdAt":"2026-01-21 16:05:48","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-8661827/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8661827/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100858834,"identity":"b6ea8d33-5e8c-4d17-b815-95a9c436f7d3","added_by":"auto","created_at":"2026-01-22 07:24:49","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":439214,"visible":true,"origin":"","legend":"","description":"","filename":"draftsgRNACD81V2merged.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8661827/v1_covered_692717a4-405b-4671-b0dc-1ee057ff0f3e.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eValidation of a CD81-based flow cytometry assay to assess dCas9 silencing activity\u003c/strong\u003e\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Inserm","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":"","lastPublishedDoi":"10.21203/rs.3.rs-8661827/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8661827/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe efficiency of CRISPR interference (CRISPRi) depends on functional dCas9 activity, yet practical and reproducible validation of dCas9-expressing cell lines remains limited. 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