{"id":423,"date":"2017-02-06T10:31:24","date_gmt":"2017-02-06T01:31:24","guid":{"rendered":"https:\/\/bionauts.jp\/?p=423"},"modified":"2025-12-16T22:38:29","modified_gmt":"2025-12-16T13:38:29","slug":"crisprcas-knock-in-method-up-to-100-efficiency","status":"publish","type":"post","link":"https:\/\/bionauts.jp\/?p=423","title":{"rendered":"CRISPR\/Cas knock-in Method up to 100% efficiency"},"content":{"rendered":"<h3>Core Benefit<\/h3>\n<ul>\n<li><strong>To produce targeted knock-in animal, such as humanized\/reporter mice\/rats model, cost and time will be saved by half compared with existing ES cell or CRSPR\/Cas9 based method.<\/strong><\/li>\n<li><strong>Enables to establish genetically modified large animals.<\/strong><\/li>\n<\/ul>\n<h3>Features<\/h3>\n<ul>\n<li>For targeted long (&gt;3Kb) cassette insertion in genome of mammalian fertilized egg with more than 50% efficiency without off-targets (published).<\/li>\n<li>Efficiency has improved to almost 100% (unpublished).<\/li>\n<li>Targeting RNAs can be easily chemically synthesized.<\/li>\n<li>No need for cell based assay to evaluate targeted digestion activity.<\/li>\n<li>Proved with EGFP(2.5kb), CreERT2 (2.5kb), flox (1kb), and other genes (&gt;3Kb), reproduced by other researchers.<\/li>\n<\/ul>\n<h3>Background and Technology<\/h3>\n<p>Even though the CRISPR\/Cas system is recognized as excellent genome editing tool, application for knock-in animal production has been limited due to low efficiency. Existing ES cell based method requires prolong period for selection from chimera origin.<\/p>\n<p>The new method is based on finding that direct injection of wild type Cas9 complex consist of three components: Cas9 protein, CRISPR RNA (crRNA), and trans activating crRNA (tracrRNA) with targeting vector can make large gene insertion with very high efficiency and accuracy.<\/p>\n<p>Immediate digestion of target DNA site and rapid degradation and deactivation of Cas9 at early one cell stage contribute to efficient knock-in and prevention of off-target and mosaicism. crRNA and tracrRNA is short enough to chemically synthesize, and Cas9 enzyme activity can be tested by cell-free test tube.<\/p>\n<p><img decoding=\"async\" loading=\"lazy\" class=\"alignnone size-full wp-image-428\" src=\"http:\/\/bionauts.jp\/wp-content\/uploads\/2017\/02\/mice.gif\" alt=\"mice\" width=\"472\" height=\"377\" \/><\/p>\n<h3>Publication and Patent<\/h3>\n<ul>\n<li>Genome Biology\u30002015\u300016:87 (DOI: 10.1186\/s13059-015-0653-x)<\/li>\n<li>WO 2016\/080097 *100% efficiency technic is not published.<\/li>\n<\/ul>\n<h3>Researcher<\/h3>\n<p>Prof. Koichi Tanaka and Associate Prof. Tomomi Aida\u3000(Tokyo Medical and Dental University, Japan)<\/p>\n<h3>Expectations<\/h3>\n<p>We are looking for companies to license-in this technology and expand its commercial applications creating genetically modified animal. Detail can be disclosed under CDA.<\/p>\n<p>Product No. WL-01183<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Achieved targeted large size gene knock-in animal production.<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_mi_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0},"categories":[1],"tags":[],"categories_of_inventions":[48],"disease_categories___indication":[],"asset_type":[78],"development_phase":[],"medicine":[],"other_than_medicine":[131,130],"material":[],"engineering":[],"software_it":[],"aioseo_notices":[],"_links":{"self":[{"href":"https:\/\/bionauts.jp\/index.php?rest_route=\/wp\/v2\/posts\/423"}],"collection":[{"href":"https:\/\/bionauts.jp\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/bionauts.jp\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/bionauts.jp\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/bionauts.jp\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=423"}],"version-history":[{"count":4,"href":"https:\/\/bionauts.jp\/index.php?rest_route=\/wp\/v2\/posts\/423\/revisions"}],"predecessor-version":[{"id":5012,"href":"https:\/\/bionauts.jp\/index.php?rest_route=\/wp\/v2\/posts\/423\/revisions\/5012"}],"wp:attachment":[{"href":"https:\/\/bionauts.jp\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=423"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/bionauts.jp\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=423"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/bionauts.jp\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=423"},{"taxonomy":"categories_of_inventions","embeddable":true,"href":"https:\/\/bionauts.jp\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories_of_inventions&post=423"},{"taxonomy":"disease_categories___indication","embeddable":true,"href":"https:\/\/bionauts.jp\/index.php?rest_route=%2Fwp%2Fv2%2Fdisease_categories___indication&post=423"},{"taxonomy":"asset_type","embeddable":true,"href":"https:\/\/bionauts.jp\/index.php?rest_route=%2Fwp%2Fv2%2Fasset_type&post=423"},{"taxonomy":"development_phase","embeddable":true,"href":"https:\/\/bionauts.jp\/index.php?rest_route=%2Fwp%2Fv2%2Fdevelopment_phase&post=423"},{"taxonomy":"medicine","embeddable":true,"href":"https:\/\/bionauts.jp\/index.php?rest_route=%2Fwp%2Fv2%2Fmedicine&post=423"},{"taxonomy":"other_than_medicine","embeddable":true,"href":"https:\/\/bionauts.jp\/index.php?rest_route=%2Fwp%2Fv2%2Fother_than_medicine&post=423"},{"taxonomy":"material","embeddable":true,"href":"https:\/\/bionauts.jp\/index.php?rest_route=%2Fwp%2Fv2%2Fmaterial&post=423"},{"taxonomy":"engineering","embeddable":true,"href":"https:\/\/bionauts.jp\/index.php?rest_route=%2Fwp%2Fv2%2Fengineering&post=423"},{"taxonomy":"software_it","embeddable":true,"href":"https:\/\/bionauts.jp\/index.php?rest_route=%2Fwp%2Fv2%2Fsoftware_it&post=423"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}