A comprehensive CRISPR/Cas9-targeted base mutagenesis identifies an amino acid substitution separating functions of SPEN in X chromosome inactivation from development

A comprehensive CRISPR/Cas9-targeted base mutagenesis identifies an amino acid substitution separating functions of SPEN in X chromosome inactivation from development | 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 comprehensive CRISPR/Cas9-targeted base mutagenesis identifies an amino acid substitution separating functions of SPEN in X chromosome inactivation from development Anton Wutz, Corinne Kaufmann, Sarah Sting This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6717927/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 While genetic screens have facilitated the dissection of protein function in animal development, advances in systematic point mutagenesis open new opportunities for forward genetics in mammalian cells. Here, we develop a CRISPR/Cas9-mediated base editing screen that allows to generate extensive maps of single amino acid substitutions of endogenous proteins. We demonstrate the application on the X-chromosomal Hprt and autosomal Msh2 gene in diploid male and haploid mouse embryonic stem cells, respectively. Finally, we use this methodology to generate a sequence-function map of the transcriptional co-repressor SPEN in X chromosome inactivation. We demonstrate that the substitution of the SPEN RRM4-residue W522 abrogates X-linked gene repression by Xist RNA and impairs H3K27-deacetylation, but does not affect Polycomb recruitment and H3K27me3 deposition. Our results demonstrate that screening in haploid cells allow the efficient identification of separation-of-function mutations that would be recessive in diploid cells suggesting applications to a wide range of areas Biological sciences/Genetics/Epigenetics Biological sciences/Genetics/Functional genomics Biological sciences/Developmental biology/Stem cells Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTable1.xlsx Supplementary Table 1 SupplementaryTable2.xlsx Supplementary Table 2 SupplementaryTable3.xlsx Supplementary Table 3 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. 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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-6717927","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":463758328,"identity":"e1deef31-502f-4dba-b241-e6617d4c626a","order_by":0,"name":"Anton 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