RECKLEEN: a lambda Red/CRISPR-Cas9 based single plasmid platform for fast, efficient, markerless, and scarless genome editing in Klebsiella pneumoniae

RECKLEEN: a lambda Red/CRISPR-Cas9 based single plasmid platform for fast, efficient, markerless, and scarless genome editing in Klebsiella pneumoniae | 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 RECKLEEN: a lambda Red/CRISPR-Cas9 based single plasmid platform for fast, efficient, markerless, and scarless genome editing in Klebsiella pneumoniae Anke Becker, Eslam Elsayed, Daniel Stukenberg, Bernd Schmeck This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5859838/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Oct, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Klebsiella pneumoniae (Kp) has evolved as a major public health threat due to its multidrug-resistance (MDR) and hypervirulence. Current genome-editing tools for Kp are constrained by cumbersome workflows, low flexibility, and limited scalability. Here, we present the RECKLEEN system —Recombineering/CRISPR-based KLebsiella Engineering for Efficient Nucleotide editing — as a single plasmid platform designed for precise genetic manipulation of Kp. RECKLEEN combines lambda Red recombineering with powerful CRISPR-Cas9-based targeted counterselection, achieving up to 99.998% killing efficiency. By implementing the near PAM-less SpG Cas9 variant in RECKLEEN, the compatible target sequence spectrum was significantly broadened. This approach enables deletions, point mutations, and DNA integrations, with efficiencies reaching 100% of the counter-selected clones. Simultaneous multi-target deletions were accomplished with up to 72% efficiency. To streamline the process, we developed a toolbox of eleven plasmids based on a modular cloning standard, enabling time- and resource-efficient assembly of editing constructs. This allows a 5-days workflow, from plasmid construction to the generation of strains with the desired genetic modification(s). The efficacy of RECKLEEN extends to various MDR Kp strains, such as ATCC 700721, ATCC BAA-1705, and ATCC 700603, demonstrating its broad applicability. RECKLEEN significantly enhances genome-editing capabilities for Kp, advancing research into its pathology and MDR mechanisms. Biological sciences/Biological techniques/Microbiology techniques Biological sciences/Microbiology/Microbial genetics/Bacterial genetics Biological sciences/Biological techniques/Molecular engineering/Synthetic biology Klebsiella pneumoniae MDR genome editing lambda Red recombineering CRISPR-Cas9 SpG Cas9 RECKLEEN Full Text Additional Declarations There is NO Competing Interest. Supplementary Files ElsayedetalSI.pdf Supplemental Material SupplementaryData.zip Supplementary Data Cite Share Download PDF Status: Published Journal Publication published 30 Oct, 2025 Read the published version in Communications Biology → 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-5859838","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":421735356,"identity":"94f41ad8-4509-4802-bf09-0e7e12f5ad24","order_by":0,"name":"Anke 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