Human cells based directed evolution of adenine base editors with improved efficiency

Human cells based directed evolution of adenine base editors with improved efficiency | 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 Human cells based directed evolution of adenine base editors with improved efficiency Junhao Fu, Qing Li, Xiaoyu Liu, Tianxiang Tu, Xiujuan Lv, Xidi Yin, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-418740/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Oct, 2021 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Adenine base editors (ABE) are novel genome-editing tools that have been harnessed to introduce precise A•T to G•C conversion in genomic DNA. However, the low activity of ABE remains a major bottleneck that precludes efficacious applications. To address this limitation, we developed a directional screening system in human cells to evolve the deaminase component of the ABE, and identified three high-activity NG-ABEmax variants: NG-ABEmax-SGK (R101S/D139G/E140K), NG-ABEmax-R (Q154R) and NG-ABEmax-K (N127K). With further engineering, we created a new, consolidated variant [NG-ABEmax-KR (N127K/Q154R)] which exhibited superior editing activity both in human cells and in mouse disease models, compared to the original NG-ABEmax. We also found that NG-ABEmax-KR efficiently introduce natural mutations in gamma globin gene promoters with more than four-fold increase in editing activity. This work provides a broadly applicable, rapidly deployable platform to directionally screen and evolve novel, user-specified traits in base editors that extend beyond augmented editing activity. Chemical Biology Epigenetics & Genomics ABE Variants Activity Directed evolution Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF. Additional Declarations Yes there is potential Competing Interest. The authors declare that they have a patent (pending) for the screening platform and the mutants in this study. Supplementary Files Supplementarydata.pdf Cite Share Download PDF Status: Published Journal Publication published 08 Oct, 2021 Read the published version in Nature Communications → 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-418740","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":25178127,"identity":"3d8d492e-ba82-4982-8b4f-ea4b1e4628be","order_by":0,"name":"Junhao Fu","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Junhao","middleName":"","lastName":"Fu","suffix":""},{"id":25178128,"identity":"ac28d5ae-796a-4724-9b97-9a3b496be77c","order_by":1,"name":"Qing Li","email":"","orcid":"https://orcid.org/0000-0002-6392-9699","institution":"State Key Laboratory of Cell 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14:11:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-418740/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-418740/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-021-26211-0","type":"published","date":"2021-10-08T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":8875267,"identity":"5f68314d-6d66-4a6c-9457-ab577924ca29","added_by":"auto","created_at":"2021-05-06 19:36:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":116745,"visible":true,"origin":"","legend":"Design of the stop-codon reversion-based screening system. \na, Different EGFP variants. Single nucleotide conversions are indicated by blue and residue substitutions are indicated by white. The stop codon was highlighted with red pentagram. b, c, Fluorescence image(b) and editing efficiencies(c) of HEK-293 cells co-transfect with EGFP WT, variant, dEGFP 1 and dEGFP 2, respectively. EGFP variant maintains the expression, while dEGFP 1 and dEGFP 2 abolishes the expression. (c). EGFP positive cells were quantified by flow cytometry. Scale bar, 10 μm. Data are mean ± s.d. of three technical replicates. d, e, The sequence of different sgRNAs (5-‘NG-3’ PAM) which were suitable for dEGFP 1 and dEGFP 2, respectively. The stop codon and target A base have been highlighted in bold and red. f, g, Editing efficiencies of A to G mutations at different positions of dEGFP 1 and dEGFP 2. Error bars indicate mean ± s.d. of three technical replicates. ","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-418740/v1/46455274706ba548c79ec620.jpg"},{"id":8875266,"identity":"738aa5b3-fecb-40ab-a97a-ca6eeb48c0c6","added_by":"auto","created_at":"2021-05-06 19:36:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":109110,"visible":true,"origin":"","legend":"Evolution of NG-ABEmax variants with improved A•T to G•C base editing activity. \na, Scheme of libraries generation design. The library 1 contain TadA* mutations and the library 2 contain TadA mutations. Red lines represent the point mutations. b, Summary of the library 1 and library 2 (A•T to G•C base editing efficiencies at A12). R represents the ratio of editing efficiency of mutants to NG-ABEmax. c, Schematic for assessing A•T to G•C base editing efficiencies of NG-ABEmax variants using the HEK293-PME cell line. Restriction sites are highlighted in red and blue. d, Agarose gel electrophoresis results of testing editing efficiency of NG-ABEmax variants with PstI. Cleaved bands from PstI are labeled with red triangle. The amplicon is 606-bp. The variants with high-activities are highlighted with red arrows. WT represents NG-ABEmax and highlighted with blue arrows. e, Boosted editing efficiencies of selected variants via EGFP-based reporter system. Error bars indicate mean ± s.d. of three technical replicates. ","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-418740/v1/992c21d871954cdfe80ae84a.jpg"},{"id":8875344,"identity":"258ebd87-1992-4958-af1e-b87cd398fdf2","added_by":"auto","created_at":"2021-05-06 19:39:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":97758,"visible":true,"origin":"","legend":"ngineering of the NG-ABEmax variants. \na, Mapping the key residues for L1-60, L1-98 and L2-156 using EGFP-based reporter system. Editing efficiencies were quantified by flow cytometry. b, Boosted editing efficiencies of NG-ABEmax-KR at endogenous genomic sites. c, Summary of boosted editing efficiencies of NG-ABEmax-KR at different A base at the EGFP sites. Error bars indicate mean ± s.d. of three technical replicates. d, e, Increased editing window of NG-ABEmax-KR in site 8 and site 10. Each A base was highlighted in red. Error bars indicate mean ± s.d. of three technical replicates. ","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-418740/v1/f6a8c3c796bf624646667ca5.jpg"},{"id":8875269,"identity":"b1c3caa2-e267-430e-8423-5d5d3523956d","added_by":"auto","created_at":"2021-05-06 19:36:49","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":80702,"visible":true,"origin":"","legend":" Application of NG-ABEmax-KR for the generation of mice disease models and human gene therapy. \na, Schematic of comparing editing activity of NG-ABEmax and NG-ABEmax-KR in mouse embryos via zygote intracytoplasmic injection. b, The newborn pups (days 10) produced by intracytoplasmic injection of NG-ABEmax or NG-ABEmax-KR mRNA and Tyr sgRNA The Tyr mutant mice (H420R) are in white and the wild-type are in black, respectively. c, Statistical analysis of on-target A-to-G base conversions induced by NG-ABEmax (n = 11), NG-ABEmaxKR (n = 20) in all pups. Data are mean ± s.d for the indicated numbers of mice. Each A base was highlighted in red. *P \u003c 0.05, ***P \u003c 0.001 by Student’s unpaired two-sided t-test d, Sanger sequence chromatograms confirmed the editing events. The desired mutation was highlighted with red star. e, Boosted editing efficiency (A7) of NG-ABEmax-KR at HBG1/2. ","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-418740/v1/a027845ad1ff14ba10021d4c.jpg"},{"id":8875083,"identity":"4ec42c24-62bf-4d4a-ac95-37f7c9045a25","added_by":"auto","created_at":"2021-05-06 19:33:49","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":106655,"visible":true,"origin":"","legend":"Molecular basis of enhanced editing efficiency of NG-ABEmax-KR. \na, Overall structural model of NG-ABEmax-KR showing SpCas9 (gray), target-strand DNA (TS, green), nontarget-strand DNA (NTS, cyan), single-target RNA (sgRNA, red), TadA (blue) and TadA* (light blue). The side chains of N127K of TadA* and Q154R of TadA are shown as red sticks. b, Modelled Interactions between key residues of NG-ABEmax-KR with TS DNA, colored as in (a). 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However, the manuscript can be downloaded and accessed as a PDF.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"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":"ABE, Variants, Activity, Directed evolution","lastPublishedDoi":"10.21203/rs.3.rs-418740/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-418740/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Adenine base editors (ABE) are novel genome-editing tools that have been harnessed to introduce precise A•T to G•C conversion in genomic DNA. However, the low activity of ABE remains a major bottleneck that precludes efficacious applications. To address this limitation, we developed a directional screening system in human cells to evolve the deaminase component of the ABE, and identified three high-activity NG-ABEmax variants: NG-ABEmax-SGK (R101S/D139G/E140K), NG-ABEmax-R (Q154R) and NG-ABEmax-K (N127K). With further engineering, we created a new, consolidated variant [NG-ABEmax-KR (N127K/Q154R)] which exhibited superior editing activity both in human cells and in mouse disease models, compared to the original NG-ABEmax. We also found that NG-ABEmax-KR efficiently introduce natural mutations in gamma globin gene promoters with more than four-fold increase in editing activity. This work provides a broadly applicable, rapidly deployable platform to directionally screen and evolve novel, user-specified traits in base editors that extend beyond augmented editing activity.","manuscriptTitle":"Human cells based directed evolution of adenine base editors with improved efficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-05-06 19:33:47","doi":"10.21203/rs.3.rs-418740/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bcc65b22-55bc-4862-a75c-9f6181dfd10e","owner":[],"postedDate":"May 6th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":4142073,"name":"Chemical Biology"},{"id":4142074,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"2021-11-22T15:32:25+00:00","versionOfRecord":{"articleIdentity":"rs-418740","link":"https://doi.org/10.1038/s41467-021-26211-0","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2021-10-08 04:00:00","publishedOnDateReadable":"October 8th, 2021"},"versionCreatedAt":"2021-05-06 19:33:47","video":"","vorDoi":"10.1038/s41467-021-26211-0","vorDoiUrl":"https://doi.org/10.1038/s41467-021-26211-0","workflowStages":[]},"version":"v1","identity":"rs-418740","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-418740","identity":"rs-418740","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}