{"id":685,"date":"2023-03-15T14:38:17","date_gmt":"2023-03-15T06:38:17","guid":{"rendered":"https:\/\/www.cellnuo.com\/?page_id=685"},"modified":"2023-04-12T13:54:26","modified_gmt":"2023-04-12T05:54:26","slug":"%e9%87%8d%e7%bb%84-cas-9","status":"publish","type":"page","link":"https:\/\/www.cellnuo.com\/?page_id=685","title":{"rendered":"\u91cd\u7ec4 Cas 9"},"content":{"rendered":"\n<p>\u91cd\u7ec4Cas 9\uff0c\u57fa\u56e0\u5de5\u7a0b\u751f\u4ea7\uff0c\u5927\u80a0\u6746\u83cc\u8868\u8fbe\u3002<\/p>\n\n\n\n<p><strong>\u4e00. \u4ea7\u54c1\u7b80\u4ecb<\/strong><strong><\/strong><\/p>\n\n\n\n<p>Cas9\u662f\u7ec6\u83cc\u548c\u53e4\u7ec6\u83cc\u5728\u957f\u671f\u6f14\u5316\u8fc7\u7a0b\u4e2d\u5f62\u6210\u7684\u4e00\u79cd\u9002\u5e94\u6027\u514d\u75ab\u9632\u5fa1\uff0c\u53ef\u5bf9\u6297\u5165\u4fb5\u7684\u75c5\u6bd2\u53ca\u5916\u6e90DNA\uff0c\u53ef\u4ee5\u5c06\u5165\u4fb5\u566c\u83cc\u4f53\u548c\u8d28\u7c92DNA\u7684\u7247\u6bb5\u6574\u5408\u5230CRISPR\u5e8f\u5217\u4e2d\uff0c\u901a\u8fc7\u76f8\u5e94\u7684CRISPR RNAs\uff08crRNAs\uff09\u6765\u6307\u5bfcCas9\u86cb\u767d\u5bf9\u540c\u6e90\u5e8f\u5217\u7684\u964d\u89e3\uff0c\u8fdb\u800c\u63d0\u4f9b\u514d\u75ab\u6027\u3002\u6839\u636eCRISPR\/Cas\u7cfb\u7edf\u7684\u7279\u6027\uff0c\u79d1\u5b66\u5bb6\u5c06\u5176\u6539\u9020\u6210\u4e86\u76ee\u524d\u6700\u9ad8\u6548\u7684\u57fa\u56e0\u7ec4\u7f16\u8f91\u5de5\u5177\u3002<\/p>\n\n\n\n<p>Cas9\u7cfb\u7edf\u7531\u4e09\u4e2a\u539f\u4ef6\u6784\u6210\uff1atracrRNA+crRNA\u5d4c\u5408\u6210\u7684sgRNA\uff08single guide RNA\uff09+\u5bbf\u4e3bDNA\u5e8f\u5217\u4e0a\u7684PAM\u5e8f\u5217\uff08protospacer adjacent motif\uff09+Cas9\u526a\u5200\uff08\u6700\u5e38\u7528\u7684\u662f\u5316\u8113\u6027\u94fe\u7403\u83cc\u6765\u6e90\u7684SpCas9\u86cb\u767d\uff09\u3002sgRNA\u5f15\u5bfcCas9\u526a\u5200\u5230\u4e0esgRNA\u76845\u2019\u672b\u7aef\u78b1\u57fa\u4e92\u8865\u7684\uff08\u957f\u7ea620nt\u7684\uff09\u9776\u5e8f\u5217\u5904\uff0c\u7d27\u63a5\u8fd9\u6bb5\u4e92\u8865\u5e8f\u5217\u4e0b\u6e38\u82e5\u5b58\u5728\u80fd\u88abCas9\u86cb\u767d\u8bc6\u522b\u7684PAM\u5e8f\u5217\uff08SpCas9\u7279\u5f02\u6027\u8bc6\u522b\u7684PAM\u662f\u75313bp\u7ec4\u6210\u7684NGG\u5e8f\u5217\uff0cN\u4ee3\u8868\u4efb\u610f\u78b1\u57fa\uff09\uff0cCas9\u526a\u5200\u5c31\u4f1a\u7528\u4e24\u4e2a\u6838\u9178\u9176\u7ed3\u6784\u57dfHNH\u548cRuvC\uff08\u57283\uff5e4nt upstream of PAM\u7684\u4f4d\u70b9\uff09\u526a\u5f00DNA\u4e24\u6761\u94fe\uff0c\u6700\u7ec8\u5f62\u6210\u53cc\u94fe\u65ad\u88c2double-strandedbreak (DSB)\u3002<\/p>\n\n\n\n<p><strong>\u4e8c. \u4ea7\u54c1\u7279\u6027<\/strong><strong><\/strong><\/p>\n\n\n\n<p>1. \u6027\u72b6 \u900f\u660e\u6db2\u4f53<\/p>\n\n\n\n<p>2. \u7eaf\u5ea6 \u7eaf\u5ea6\u226590%\uff08SDS-PAGE\/SEC-HPLC\uff09<\/p>\n\n\n\n<p>3. \u5bbf\u4e3b\u86cb\u767d\u8d28\u6b8b\u7559\u91cf \u5c0f\u4e8e100ng\/mg<\/p>\n\n\n\n<p>4. \u7ec6\u83cc\u5185\u6bd2\u7d20 \u5c0f\u4e8e10EU\/mg<\/p>\n\n\n\n<p>5. \u5bbf\u4e3bDNA\u6b8b\u7559\u91cf \u5c0f\u4e8e10ng\/mg<\/p>\n\n\n\n<p>6. DNA\u9176\u6b8b\u7559 \u9634\u6027<\/p>\n\n\n\n<p>7. RNA\u9176\u6b8b\u7559 \u9634\u6027<\/p>\n\n\n\n<p>8. \u5916\u5207\u9176\u6d3b\u6027 \u5927\u4e8e\u7b49\u4e8e80%<\/p>\n\n\n\n<p>9. \u6d53\u5ea6 \u5927\u4e8e\u7b49\u4e8e5mg\uff08ABS\uff09<strong><\/strong><\/p>\n\n\n\n<p><strong>\u4e09. \u4ea7\u54c1\u5e94\u7528<\/strong><strong><\/strong><\/p>\n\n\n\n<p><strong>\u57fa\u56e0\u6572\u9664\uff08Knock-out\uff09 \u7b80\u79f0KO<\/strong><strong><\/strong><\/p>\n\n\n\n<p>Cas9\u86cb\u767d\u53ef\u4ee5\u5bf9\u9776\u57fa\u56e0\u7ec4\u8fdb\u884c\u526a\u5207\uff0c\u5f62\u6210DNA\u7684\u53cc\u94fe\u65ad\u88c2\u3002\u5728\u901a\u5e38\u60c5\u51b5\u4e0b\uff0c\u7ec6\u80de\u4f1a\u91c7\u7528\u9ad8\u6548\u7684\u975e\u540c\u6e90\u672b\u7aef\u8fde\u63a5\u65b9\u5f0f\u5bf9\u65ad\u88c2\u7684DNA\u8fdb\u884c\u4fee\u590d\u3002\u4f46\u662f\uff0c\u5728\u4fee\u590d\u8fc7\u7a0b\u4e2d\u901a\u5e38\u4f1a\u53d1\u751f\u78b1\u57fa\u63d2\u5165\u6216\u7f3a\u5931\u7684\u9519\u914d\u73b0\u8c61\uff0c\u9020\u6210\u79fb\u7801\u7a81\u53d8\u4f7f\u9776\u6807\u57fa\u56e0\u5931\u53bb\u529f\u80fd\uff0c\u4ece\u800c\u5b9e\u73b0\u57fa\u56e0\u6572\u9664\u3002\u4e3a\u4e86\u63d0\u9ad8CRISPR\u7cfb\u7edf\u7684\u7279\u5f02\u6027\uff0c\u53ef\u5c06Cas9\u7684\u4e00\u4e2a\u7ed3\u6784\u57df\u8fdb\u884c\u7a81\u53d8\uff0c\u5f62\u6210\u53ea\u80fd\u5bf9DNA\u5355\u94fe\u8fdb\u884c\u5207\u5272\u9020\u6210DNA\u7f3a\u53e3\u7684Cas9\u6838\u9178\u9176\uff0c\u60f3\u8981\u5f62\u6210\u53cc\u94fe\u65ad\u88c2\u7684\u6548\u679c\u53ef\u4ee5\u8bbe\u8ba1\u4e24\u6761sgRNA\u5e8f\u5217\uff0c\u4e24\u6761sgRNA\u7279\u5f02\u6027\u7684\u9776\u5411\u7ed3\u5408DNA\u4e92\u8865\u7684\u4e24\u6761\u94fe\u7684\u9776\u6807\u5e8f\u5217\uff0c\u5373\u53ef\u5f62\u6210DNA\u65ad\u88c2\uff0c\u5e76\u5728\u4fee\u590d\u8fc7\u7a0b\u4e2d\u901a\u8fc7\u79fb\u7801\u7a81\u53d8\u5b9e\u73b0\u57fa\u56e0\u6572\u9664\u3002<\/p>\n\n\n\n<p><strong>\u57fa\u56e0\u6572\u5165\uff08Knock-in\uff09 \u7b80\u79f0KI<\/strong><strong><\/strong><\/p>\n\n\n\n<p>\u5f53DNA\u53cc\u94fe\u65ad\u88c2\u540e\uff0c\u5982\u679c\u6709DNA\u4fee\u590d\u6a21\u677f\u8fdb\u5165\u5230\u7ec6\u80de\u4e2d\uff0c\u57fa\u56e0\u7ec4\u65ad\u88c2\u90e8\u5206\u4f1a\u4f9d\u636e\u4fee\u590d\u6a21\u677f\u8fdb\u884c\u540c\u6e90\u91cd\u7ec4\u4fee\u590d\uff08HDR\uff09\uff0c\u4ece\u800c\u5b9e\u73b0\u57fa\u56e0\u6572\u5165\u3002HDR\u4fee\u590d\u6a21\u5f0f\u5728\u7ec6\u80de\u4e2d\u53d1\u751f\u7387\u8f83\u4f4e\uff0c\u901a\u5e38\u5c0f\u4e8e10%\u3002\u4e3a\u4e86\u589e\u52a0\u57fa\u56e0\u6572\u5165\u7684\u6210\u529f\u7387\uff0c\u76ee\u524d\u6709\u5f88\u591a\u79d1\u5b66\u5bb6\u81f4\u529b\u4e8e\u63d0\u9ad8HDR\u6548\u7387\uff0c\u5c06\u7f16\u8f91\u7684\u7ec6\u80de\u540c\u6b65\u81f3HDR\u6700\u6d3b\u8dc3\u7684\u7ec6\u80de\u5206\u88c2\u65f6\u671f\uff0c\u4fc3\u8fdb\u4fee\u590d\u65b9\u5f0f\u4ee5HDR\u8fdb\u884c\uff1b\u6216\u8005\u5229\u7528\u5316\u5b66\u65b9\u6cd5\u6291\u5236\u57fa\u56e0\u8fdb\u884cNHEJ\uff0c\u63d0\u9ad8HDR\u7684\u6548\u7387\u3002<\/p>\n\n\n\n<p><strong>\u591a\u91cd\u7f16\u8f91\uff08Multiplex Editing\uff09<\/strong><strong><\/strong><\/p>\n\n\n\n<p>\u5c06\u591a\u4e2asgRNA\u8d28\u7c92\u8f6c\u5165\u5230\u7ec6\u80de\u4e2d\uff0c\u53ef\u540c\u65f6\u5bf9\u591a\u4e2a\u57fa\u56e0\u8fdb\u884c\u7f16\u8f91\uff0c\u5177\u6709\u57fa\u56e0\u7ec4\u529f\u80fd\u7b5b\u9009\u4f5c\u7528\u3002\u591a\u91cd\u7f16\u8f91\u7684\u5e94\u7528\u5305\u62ec\uff1a\u4f7f\u7528\u53ccCas9 nickases\u63d0\u9ad8\u57fa\u56e0\u6572\u9664\u7684\u51c6\u786e\u7387\u3001\u5927\u8303\u56f4\u7684\u57fa\u56e0\u7ec4\u7f3a\u5931\u53ca\u540c\u65f6\u7f16\u8f91\u4e0d\u540c\u7684\u57fa\u56e0\u3002\u901a\u5e38\u60c5\u51b5\u4e0b\uff0c\u4e00\u4e2a\u8d28\u7c92\u4e0a\u53ef\u4ee5\u6784\u5efa2\uff5e7\u4e2a\u4e0d\u540c\u7684sgRNA\u8fdb\u884c\u591a\u91cdCRISPR\u57fa\u56e0\u7f16\u8f91\u3002<\/p>\n\n\n\n<p><strong>\u56db. \u50a8\u5b58\u548c\u8fd0\u8f93\u7a33\u5b9a\u6027<\/strong><strong><\/strong><\/p>\n\n\n\n<p>\u50a8\u5b58\u7a33\u5b9a\u6027\uff1a-25\u81f3-15\u2103\u6e29\u5ea6\u4e0b\uff0c\u4e24\u5e74\u6709\u6548\u3002<\/p>\n\n\n\n<p>\u8fd0\u8f93\u7a33\u5b9a\u6027\uff1a\u51b7\u85cf\u8fd0\u8f93\uff0c\u6d3b\u6027\u7a33\u5b9a\u3002<\/p>\n\n\n\n<p>\u6ce8\u610f\uff1a\u672c\u4ea7\u54c1\u4e0d\u5efa\u8bae-80\u2103\u4fdd\u5b58\uff0c\u53cd\u590d\u51bb\u878d\u4f1a\u964d\u4f4e\u672c\u4ea7\u54c1\u7684\u9176\u6d3b\u6027\u3002<\/p>\n\n\n\n<p><strong>\u4e94. \u4ea7\u54c1\u4f18\u52bf<\/strong><strong><\/strong><\/p>\n\n\n\n<p>\u65e0\u52a8\u7269\u6e90\u6027\uff1a\u91cd\u7ec4\u751f\u4ea7\uff0c\u65e0\u5916\u6e90\u6027\u7684\u75c5\u6bd2\u6c61\u67d3\uff0c\u751f\u4ea7\u8fc7\u7a0b\u4e0d\u4f7f\u7528\u4efb\u4f55\u52a8\u7269\u6e90\u539f\u6599\u3002<\/p>\n\n\n\n<p>\u8d28\u91cf\u7a33\u5b9a\uff1a\u6279\u91cf\u751f\u4ea7\uff08\u7a33\u5b9a\u751f\u4ea7\u4f9b\u5e94100g\uff09\uff0c\u53ef\u4fdd\u8bc1\u7a33\u5b9a\u8fde\u7eed\u7684\u6279\u6b21\u751f\u4ea7\uff1b\u4ea7\u54c1\u6279\u6b21\u95f4\u65e0\u5dee\u5f02\uff0c\u8d28\u91cf\u7a33\u5b9a\u3002<\/p>\n\n\n\n<p>\u7eaf\u5ea6\u9ad8\uff1a\u6bd4\u6d3b\u9ad8\uff1b\u5bbf\u4e3b\u86cb\u767d\u6b8b\u7559\u5c0f\u4e8e\u751f\u7269\u5236\u54c1\u9650\u5ea6\u8981\u6c42\u3002<\/p>\n\n\n\n<p>\u7b26\u5408\u6cd5\u89c4\u8981\u6c42\uff1a\u751f\u4ea7\u8bbe\u5907\u548c\u751f\u4ea7\u73af\u5883\u7b26\u5408\u76f8\u5173\u6cd5\u89c4\u8981\u6c42\uff0c\u751f\u4ea7\u8fc7\u7a0b\u5b8c\u5168\u7b26\u5408GMP\u6307\u5bfc\u539f\u5219\u3002<\/p>\n\n\n\n<p>\u8d28\u91cf\u6587\u4ef6\u5b8c\u6574\uff1a\u6309\u5ba2\u6237\u9700\u6c42\uff0c\u53ef\u63d0\u4f9b\u76f8\u5173\u6cd5\u89c4\u652f\u6301\u6587\u4ef6\u3002<\/p>\n\n\n\n<p><strong>\u516d. \u76f8\u5173\u4ea7\u54c1<\/strong><strong><\/strong><\/p>\n\n\n\n<p>\u91cd\u7ec4sumo\u9176\uff1b<\/p>\n\n\n\n<p>\u91cd\u7ec4TEV\u9176\uff1b<\/p>\n\n\n\n<p>\u91cd\u7ec4\u80a0\u6fc0\u9176\u3002<\/p>\n\n\n\n<p>\u4ec5\u4f9b\u79d1\u7814\u751f\u4ea7\u4f7f\u7528\uff0c\u4e0d\u80fd\u7528\u4e8e\u4eba\u4f53\u3002<\/p>\n","protected":false},"excerpt":{"rendered":"<p>\u91cd\u7ec4Cas 9\uff0c\u57fa\u56e0\u5de5\u7a0b\u751f\u4ea7\uff0c\u5927\u80a0\u6746\u83cc\u8868\u8fbe\u3002 \u4e00. \u4ea7\u54c1\u7b80\u4ecb Cas9\u662f\u7ec6\u83cc\u548c\u53e4\u7ec6\u83cc\u5728\u957f\u671f\u6f14\u5316\u8fc7\u7a0b\u4e2d\u5f62\u6210\u7684\u4e00\u79cd [&hellip;]<\/p>\n<p class=\"more-link\"><a href=\"https:\/\/www.cellnuo.com\/?page_id=685\"  class=\"themebutton\">Read More<\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"categories":[],"tags":[],"class_list":["post-685","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/www.cellnuo.com\/index.php?rest_route=\/wp\/v2\/pages\/685","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.cellnuo.com\/index.php?rest_route=\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.cellnuo.com\/index.php?rest_route=\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.cellnuo.com\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.cellnuo.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=685"}],"version-history":[{"count":2,"href":"https:\/\/www.cellnuo.com\/index.php?rest_route=\/wp\/v2\/pages\/685\/revisions"}],"predecessor-version":[{"id":738,"href":"https:\/\/www.cellnuo.com\/index.php?rest_route=\/wp\/v2\/pages\/685\/revisions\/738"}],"wp:attachment":[{"href":"https:\/\/www.cellnuo.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=685"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.cellnuo.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=685"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.cellnuo.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=685"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}