A Unique Phage DUF669 Protein mediates Post-cleavage Repair Against Host CRISPR Immunity

A Unique Phage DUF669 Protein mediates Post-cleavage Repair Against Host CRISPR Immunity | 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 A Unique Phage DUF669 Protein mediates Post-cleavage Repair Against Host CRISPR Immunity Yingfei Ma, Heng zhu, Shengkun Dai, Hewen Deng, Shifeng Hou, Yuelong Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8337580/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 Phages have evolved various anti-CRISPR(Acr) proteins evade hosts’ immunity by direct CRISPR interference. However, whether other counter-CRISPR mechanisms exist remains unexplored. Here, we report a phage-encoded two-protein system, Healer, which neutralizes CRISPR immunity via a post-cleavage DNA repair mechanism. This phage replication-associated system comprises two proteins: Gp63 (a DUF669 domain-containing protein), and Gp64 (a AAA domain). Mechanistic investigations elucidate that Gp63 acts as a rapid-response effector of CRISPR-induced DNA break, following the binding of ssDNA allows the Gp64 to mediate homologous recombination, repairing CRISPR-induced phage DNA break and enabling phage survival. Notably, co-expression Healer system with CRISPR-Cas9/Cas12 in E. coli, P. aeruginosa, and A. baumannii, demonstrated higher phage genome-editing efficiency. Conclusively, our findings represent a vital anti-CRISPR complementary strategy, providing a promising tool for genome manipulation. Biological sciences/Microbiology/Virology Biological sciences/Molecular biology/DNA damage and repair Phage counter-defense Homologous recombination DUF669 domain Single-strand DNA-binding protein (SSB) CRISPR-Cas immunity Genome editing Full Text Additional Declarations There is NO Competing Interest. Supplementary Files extendeddata.rar The original files predicted by AlphaFold 3 and the phage sequences supplementaryvideoS1.gif The complex structure of Csy-dsDNA-Gp63 predicted by AlphaFold3 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. 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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-8337580","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":568906813,"identity":"cf479a4a-e46b-4c13-ad33-6425b8382318","order_by":0,"name":"Yingfei 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