Highly efficient generation of germline mutations using CRISPR/Cas9 in the speckled wood butterfly Pararge aegeria

Highly efficient generation of germline mutations using CRISPR/Cas9 in the speckled wood butterfly Pararge aegeria | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Ecology and Evolution This is a preprint and has not been peer reviewed. Data may be preliminary. 14 January 2025 V1 Latest version Share on Highly efficient generation of germline mutations using CRISPR/Cas9 in the speckled wood butterfly Pararge aegeria Authors : Anna Shoshan 0009-0001-1406-4254 [email protected] , Kalle Tunström , Christopher Wheat 0000-0003-1863-2340 , and Karl Gotthard 0000-0002-4560-6271 Authors Info & Affiliations https://doi.org/10.22541/au.173685827.71796116/v1 Published Ecology and Evolution Version of record Peer review timeline 347 views 195 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract To date, the use of CRISPR/Cas9 technology in ecological-model species for validating genotype to phenotype connections has focused primarily on visual phenotypes using G0 mutations coupled with analyses of resulting mosaic phenotypes. However, studies of physiological phenotypes necessitate germline mutations in order to assess non-visible phenotypic effects, thus dedicated efforts to developing efficient germline mutations in ecological model species are needed. Here we applied the CRISPR/Cas9 technology to an ecological model species, the speckled wood butterfly (Pararge aegeria). We targeted yellow-y, which is required for the production of black melanin, as yellow-y loss of function (LOF) mutations are not lethal and easy to phenotype, affording efficient assessment of F0 and germline mutations. To explore what factors may affect efficiency of transformation, we employed four alternative treatments, including variation in sgRNAs and their concentrations. Color changes in the head capsule of first larval instar as well as adult wing color were used as indicators of successful knockouts. Individuals with wings that were at least 50% transformed were mated, with their F1 offspring assessed for the presence of germline mutations. Our CRISPR/Cas9 technique was highly efficient at generating LOF mutations in yellow-y. Across all treatments, nearly 80% of adults exhibited mosaic LOF phenotypes, of which nearly 30% appeared to have 100% LOF phenotypes. Crosses between adults exhibiting at least 50% LOF phenotypes resulted in fully transformed offspring, revealing high incidence of germline LOF mutations in yellow-y. We provide a detailed protocol on how to obtain high germline LOF mutation efficiency in order to advance the study of genotype-phenotype connections for non-visible physiological traits across natural populations of this and other model ecological species. Supplementary Material File (figure 3.pdf) Download 21.62 MB File (main document.docx) Download 271.11 KB Information & Authors Information Version history V1 Version 1 14 January 2025 Peer review timeline Published Ecology and Evolution Version of Record 29 Jun 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Ecology and Evolution Keywords genetics invertebrate laboratory method development terrestrial Authors Affiliations Anna Shoshan 0009-0001-1406-4254 [email protected] Stockholms University Department of Zoology View all articles by this author Kalle Tunström Lund University View all articles by this author Christopher Wheat 0000-0003-1863-2340 Stockholm University View all articles by this author Karl Gotthard 0000-0002-4560-6271 Stockholm University View all articles by this author Metrics & Citations Metrics Article Usage 347 views 195 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Anna Shoshan, Kalle Tunström, Christopher Wheat, et al. Highly efficient generation of germline mutations using CRISPR/Cas9 in the speckled wood butterfly Pararge aegeria. Authorea . 14 January 2025. DOI: https://doi.org/10.22541/au.173685827.71796116/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.173685827.71796116/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'9fe9eb44ea061640',t:'MTc3OTI2NTE5Mw=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();