Switched Fluorogenic CRISPR-tDeg System for RNA Imaging in Living Cells

Switched Fluorogenic CRISPR-tDeg System for RNA Imaging in Living Cells | 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 Switched Fluorogenic CRISPR-tDeg System for RNA Imaging in Living Cells Minghai Chen, Huimin Zhong, Jiayan Zhou, Fujun Qin, Xian-En Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6073102/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 RNA displays significant heterogeneity in its structures, dynamics, and functional roles within cells. CRISPR-based imaging methods provide a powerful approach to visualize RNA molecules in living cells. However, the expression of constitutive fluorescence modules in traditional CRISPR-based tools always produce high background and nonspecific signals. To address these challenges, we developed a switched fluorescent CRISPR-tDeg (CtDeg) system that minimizes background for RNA imaging in living cells. This CtDeg system consists of a signal module-deactivated Cas13 protein (dCas13)-degron complex and an engineered switched sgRNA. The signal module-fused complex is degraded unless it binds to the engineered sgRNA, which is activated upon targeting the desired RNA. This interaction will generate a stable signal module-dCas13: sgRNA complex, enabling specific imaging of RNA in living cells. Using the CtDeg system, we visualized the movement and assembly dynamics of paraspeckles at the RNA level. Additionally, we tracked SARS-CoV-2 genome dynamics at the early stage after viral invasion for the first time, providing direct imaging data that demonstrate SARS-CoV-2 infection up-regulates the expression levels of the disease-associated NEAT1 gene, thereby filling a gap in this research area. The CtDeg system represents a powerful tool for specific imaging of RNA to reveal the underlying functions and interactions within living cells. Biological sciences/Biochemistry/RNA Biological sciences/Biological techniques/Imaging Biological sciences/Chemical biology/RNA Biological sciences/Molecular biology CtDeg dCas13 RNA imaging low background SARS-CoV-2 living cells Full Text Additional Declarations There is NO Competing Interest. Supplementary Files VideoS1.avi Video S1 VideoS2.avi Video S2 VideoS3.avi Video S3 SupportingInformation.pdf Supporting Information 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-6073102","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":425427792,"identity":"77bdc619-e168-41d6-a470-a0021b98a571","order_by":0,"name":"Minghai 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