Strategies for enhancing efficient generation of mice via CRISPR/HDR-mediated knock-in

Strategies for enhancing efficient generation of mice via CRISPR/HDR-mediated knock-in | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Strategies for enhancing efficient generation of mice via CRISPR/HDR-mediated knock-in Saumya Mary Mathew This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1687332/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Jan, 2023 Read the published version in Molecular Biology Reports → Version 1 posted 4 You are reading this latest preprint version Abstract CRISPR/Cas9 framework is generally used to generate genetically modified mouse models. The CRISPR (clustered regularly interspaced short palindromic repeat) gene editing technique, can efficiently generate knock-outs using the non-homologous end-joining (NHEJ) repair pathway. Small knock-ins also work precisely using a repair template with help of homology-directed-repair (HDR) mechanism. However, when the fragment size is larger than 4- 5 kb, the knock-in tends to be error prone and the efficiency decreases. Certain types of modifications, in particular insertions of very large DNA fragments(10–100kb) or entire gene replacements, are still difficult. The HDR process needs further streamlining and improvement. Here in this review, we describe methods to enhance the efficiency of the knock-in through checking each step from the guide design to the microinjection and choice of the oocyte donors. This helps in understanding the parameters that can be modified to get improved knock-in efficiency via CRISPR targeting. CRISPR HDR Knock-in efficiency transgenic mice microinjection Full Text Cite Share Download PDF Status: Published Journal Publication published 26 Jan, 2023 Read the published version in Molecular Biology Reports → Version 1 posted Reviewers agreed at journal 31 May, 2022 Reviewers invited by journal 24 May, 2022 Editor assigned by journal 24 May, 2022 First submitted to journal 23 May, 2022 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 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-1687332","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":108507856,"identity":"2a4ae609-e323-4e3e-a311-e40607416733","order_by":0,"name":"Saumya Mary Mathew","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYFADCSD+YCAB5RkQoYMHqJhxBslamHmIcY58+xnTzTw12+TtpZuPSdsUWNjzNzA//MBQcAenFoMzOWa3eY7dNuyROZYmnWMgkTjjAJuxBIPBM9xaGEBa2G4z9kgAGUAtCUBfmAHFD+N2WP8boJZ/t+17JPK/3bYwkLA3YGD/hlcLww2g4bxttxOBtrDdZjCQYNzAwIPfFoMbz8puzu27ndxzI838Zw/IL4d5ioHOw+ew5G033ny7bds+I/mxwY8/dfb87e0bP3z4g8dhQMCEGh3MQJyAVwMw0n8QUDAKRsEoGAUjHAAARKxQGEzz45UAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1452-605X","institution":"Indian Institute of Science","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Saumya","middleName":"Mary","lastName":"Mathew","suffix":""}],"badges":[],"createdAt":"2022-05-24 06:34:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1687332/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1687332/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-023-08278-8","type":"published","date":"2023-01-26T18:33:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":21964618,"identity":"d3d772bc-a14f-436d-9a1f-93e2c6c48756","added_by":"auto","created_at":"2022-05-27 14:29:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":565068,"visible":true,"origin":"","legend":"","description":"","filename":"MOLED2202763reviewer1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1687332/v1_covered.pdf"}],"financialInterests":"","formattedTitle":"Strategies for enhancing efficient generation of mice via CRISPR/HDR-mediated knock-in","fulltext":[{"header":"Full Text","content":"This preprint is available for \u003ca href='/article/rs-1687332/latest.pdf' target='_blank'\u003edownload as a PDF\u003c/a\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"CRISPR, HDR, Knock-in efficiency, transgenic mice, microinjection","lastPublishedDoi":"10.21203/rs.3.rs-1687332/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1687332/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"CRISPR/Cas9 framework is generally used to generate genetically modified mouse models. 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