{"id":36225,"date":"2021-03-16T00:58:12","date_gmt":"2021-03-15T16:58:12","guid":{"rendered":"https:\/\/case.ntu.edu.tw\/blog\/?p=36225"},"modified":"2021-02-23T11:11:51","modified_gmt":"2021-02-23T03:11:51","slug":"%e5%9f%ba%e5%9b%a0%e7%b7%a8%e8%bc%af%e6%8a%80%e8%a1%93crispr%e5%92%8ctalen%e7%9a%84%e6%af%94%e8%bc%83","status":"publish","type":"post","link":"https:\/\/case.ntu.edu.tw\/blog\/?p=36225","title":{"rendered":"\u57fa\u56e0\u7de8\u8f2f\u6280\u8853CRISPR\u548cTALEN\u7684\u6bd4\u8f03"},"content":{"rendered":"<p><span style=\"color: #333399;\"><strong>CRISPR\u662f\u9769\u547d\u6027\u7684\u57fa\u56e0\u7de8\u8f2f\u6280\u8853\uff0c\u53ef\u5354\u52a9\u4eba\u985e\u6539\u5bebDNA\u7de8\u78bc\uff0c\u61c9\u7528\u7bc4\u570d\u5341\u5206\u5ee3\u6cdb\uff0c\u5305\u62ec\u8fb2\u7522\u54c1\u3001\u98df\u54c1\u3001\u74b0\u4fdd\u53ca\u751f\u91ab\u7b49\u9818\u57df\u3002\u53e6\u4e00\u7a2e\u57fa\u56e0\u7de8\u8f2f\u6280\u8853TALEN\u662f\u7d93\u4eba\u5de5\u6539\u9020\u7684\u9650\u5236\u6027\u5167\u5207\u9176\uff0c\u53ef\u5c0dDNA\u4f4d\u9ede\u9032\u884c\u8fa8\u8b58\u3001\u6a19\u9776\u8207\u7de8\u8f2f\uff0c\u8b58\u5225DNA\u5e8f\u5217\u7684\u80fd\u529b\u8207Cas9\u76f8\u4f3c\uff0c\u4f46\u4e8c\u8005\u4f5c\u7528\u65b9\u5f0f\u4e0d\u540c\u3002\u8fd1\u5e74\uff0c\u5168\u7403\u79d1\u5b78\u5bb6\u70ba\u589e\u52a0\u57fa\u56e0\u7de8\u8f2f\u7684\u7cbe\u6e96\u5ea6\u8207\u6548\u7387\uff0c\u5df2\u6295\u5165\u4e86\u5927\u91cf\u6642\u9593\u3001\u4eba\u529b\u53ca\u8cc7\u672c\uff0c\u8a55\u4f30\u9664CRISPR\u4ee5\u5916\u5176\u4ed6\u57fa\u56e0\u7de8\u8f2f\u5de5\u5177\u7684\u53ef\u884c\u6027\uff0c\u6211\u5011\u5728\u672c\u6587\u5c07\u7c21\u4ecbCRISPR\u53caTALEN\u5169\u7a2e\u57fa\u56e0\u7de8\u8f2f\u6280\u8853\u7684\u7279\u9ede\uff0c\u4e26\u5c0d\u5176\u4f5c\u6bd4\u8f03\u3002<\/strong><\/span><\/p>\n<p><strong>\u64b0\u6587\uff5c\u9673\u6df5\u9293<\/strong><\/p>\n<figure id=\"attachment_36229\" aria-describedby=\"caption-attachment-36229\" style=\"width: 700px\" class=\"wp-caption aligncenter\"><img fetchpriority=\"high\" decoding=\"async\" class=\"wp-image-36229\" src=\"http:\/\/case.ntu.edu.tw\/blog\/wp-content\/uploads\/2021\/02\/65464.jpg\" alt=\"\" width=\"700\" height=\"833\" srcset=\"https:\/\/case.ntu.edu.tw\/blog\/wp-content\/uploads\/2021\/02\/65464.jpg 1172w, https:\/\/case.ntu.edu.tw\/blog\/wp-content\/uploads\/2021\/02\/65464-252x300.jpg 252w, https:\/\/case.ntu.edu.tw\/blog\/wp-content\/uploads\/2021\/02\/65464-768x913.jpg 768w, https:\/\/case.ntu.edu.tw\/blog\/wp-content\/uploads\/2021\/02\/65464-861x1024.jpg 861w\" sizes=\"(max-width: 700px) 100vw, 700px\" \/><figcaption id=\"caption-attachment-36229\" class=\"wp-caption-text\">\u5716\u7247\u4f86\u6e90: Introductory Chapter: Gene Editing Technologies and Applications. In Gene Editing-Technologies and Applications, IntechOpen, 2019.<\/figcaption><\/figure>\n<p>&nbsp;<\/p>\n<p><span style=\"color: #008080;\"><strong>\u25cfCRISPR<\/strong><strong>\u7684\u7279\u9ede<\/strong><\/span><\/p>\n<p>\u5916\u4f86\u7684\u8cea\u9ad4\u6216\u75c5\u6bd2\uff08\u566c\u83cc\u9ad4\uff09DNA\u9996\u6b21\u9032\u5165\u7d30\u83cc\u5f8c\u4e26\u672a\u88ab\u5b8c\u5168\u5206\u89e3\uff0c\u7d93\u52a0\u5de5\u5f8c\u53ef\u5d4c\u5165\u7d30\u83cc\u57fa\u56e0\u9ad4\u800c\u88ab\u7a31\u70ba CRISPR \u9663\u5217\uff08array\uff09\uff0c\u6b64\u7279\u6b8a\u5340\u6bb5\u80fd\u5920\u8f49\u9304\u8a0a\u606f RNA\uff08messenger RNA, mRNA\uff09\uff0c\u800c\u5177\u6709\u5207\u5272 DNA \u6d3b\u6027\u7684 Cas9 \u86cb\u767d\uff08\u9650\u5236\u5167\u5207\u9176\uff09\u5247\u6703\u5229\u7528 mRNA \u7247\u6bb5\u53bb\u8fa8\u8a8d\u4e92\u88dc\u6027\u7684 DNA \u7247\u6bb5\uff0c\u4e26\u5207\u9664\u7b26\u5408\u5e8f\u5217\u7684\u6a19\u7684\u7269\u3002\u55ae\u80a1\u5c0e\u5f15 RNA \uff08single-guiding RNA, sgRNA\uff09\u6703\u8207\u5bbf\u4e3b\u7279\u5b9a\u7684 DNA\u5e8f\u5217\u53caCas 9\u86cb\u767d\u7d50\u5408\uff0c\u5176\u4e2d sg RNA \u7684 protospacer \u5e8f\u5217\uff0817\uff0d20 \u500b\u6838\u82f7\u9178\uff09\u8ca0\u8cac\u8fa8\u8b58\u5c08\u4e00\u6027\u7684 DNA \u5e8f\u5217\uff0cCas9 \u86cb\u767d\u8cea\u5247\u6703\u8fa8\u8a8d protospacer \u5e8f\u5217\u9130\u8fd1\u7684\u5340\u57df\uff08protospacer adjacent motif, PAM\uff09\u4e0a\u6e38 3 \u500b\u6838\u82f7\u9178\u7684\u4f4d\u9ede\uff0c\u5229\u7528\u6838\u9178\u5167\u5207\u9176\u4f7f\u6a19\u7684 DNA \u7522\u751f\u96d9\u80a1\u65b7\u88c2\uff08double-stranded break, DSB\uff09\u3002\u56e0\u6b64\uff0c\u4fee\u6539 sgRNA \u5e8f\u5217\uff0c\u5373\u53ef\u6539\u8b8a Cas9 \u86cb\u767d\u7684\u5c08\u4e00\u6027\uff0c\u4f7f\u5176\u88c1\u5207\u53e6\u4e00\u4e0d\u540c\u5e8f\u5217\u7684 DNA\u3002\u8207\u50b3\u7d71\u7684\u57fa\u56e0\u7de8\u8f2f\u6280\u8853\u76f8\u8f03\uff0cCRISPR \u73fe\u4eca\u53d7\u5230\u6700\u5ee3\u6cdb\u63a1\u7528\uff0c\u4e3b\u8981\u662f\u56e0\u5176\u540c\u6642\u5177\u6709\u4e0b\u5217\u512a\u9ede\uff1a<\/p>\n<p>1.\u7121\u7269\u7a2e\u9650\u5236\uff1a\u52d5\u7269\u3001\u690d\u7269\u53ca\u5fae\u751f\u7269\u7b49\u751f\u7269\u5747\u9069\u7528\u6b64\u6280\u8853\u9032\ufa08\u57fa\u56e0\u7de8\u8b6f\u3002<\/p>\n<p>2.\u7c21\u55ae\uff1a\u57fa\u56e0\u6a19\u9776\u9ede\uff08target site\uff09\u641c\u5c0b\u5bb9\uf9e0\uff0cPAM\u4f4d\u9ede (NGG) \u5e73\u5747\u65bc128 \u9e7c\u57fa\u5c0d\uff08base pair, bp\uff09\u96a8\u6a5f DNA \u5e8f\uf99c\u4e2d\u5c31\u6703\u51fa\u73fe\u4e00\u6b21\uff0c\u4e14DNA\u6b63\u53cd\uf978\u80a1\u7686\u53ef\u8a2d\u8a08\uff0c\u8cea\u9ad4\u69cb\u7bc9\u5931\u6557\uf961\u4f4e\uff0cCas9\u86cb\u767d\u8fa8\uf9fc\u6548\uf961\u9ad8\u3002<\/p>\n<p>3.\u6e96\u78ba\uff1a\u5229\u7528RNA\u8207DNA\u4e92\u88dc\u6027\u9e7c\u57fa\u914d\u5c0d\uff08complementary base pairing\uff09\u7684\u539f\u7406\uff0csgRNA\u53ef\u4ee5\u6b63\u78ba\u7684\u8fa8\u8a8d\u6a19\u7684DNA\u5e8f\u5217\uff0c\u9084\u53ef\u540c\u6642\u653e\u51652\u500b\u4ee5\u4e0a\u7684sgRNA \uf92d\u9054\u6210\u540c\u6642\u5254\u9664\u591a\u500b\u57fa\u56e0\u7684\u76ee\u7684\u3002<\/p>\n<p>4.\u8fc5\u901f\uff1a\u4f7f\u7528\u6642\u50c5\u9700\u5408\u6210\u7279\u5b9a\u5e8f\u5217\u7684DNA\uff0c\u8f03\u88fd\u9020\u91cd\u7d44\u86cb\u767d\u66f4\u70ba\u7a69\u5b9a\u4e14\u8f03\u6709\u6548\u7387\uff1b\u96d9\u5408\u4e00\u6216\u4e09\u5408\u4e00\u8f09\u9ad4\u7c21\u5316\u7d30\u80de\u8f49\u67d3\uff08transfection\uff09\u7684\u96e3\ufa01\uff0c\u518d\u914d\u5408\u5831\u5c0e\u57fa\u56e0\uff08reporter gene\uff09\u7684\u8868\u73fe\uff0c\u5982\u87a2\u5149\u86cb\u767d\u6216\u6297\u751f\u7d20\u7be9\u9078\u57fa\u56e0\uff0c\u53ef\u4f7f\u7be9\u9078\uf901\u52a0\u8fc5\u901f\u3002<\/p>\n<p>5.\u7d93\u6fdf\uff1a\u88fd\u4f5c\u6750\u6599\u50c5\u6709\u5f15\u5b50\uff08primer\uff09\u3001\u8cea\u9ad4\u53ca\u7c21\u55ae\u7684\u9175\u7d20\uff0c\u6240\u9700\u6210\u672c\u8f03\u50b3\u7d71\u65b9\u6cd5\u70ba\u4f4e\u3002<\/p>\n<p><span style=\"color: #008080;\"><strong>\u25cfTALEN<\/strong><strong>\u7684\u7279\u9ede<\/strong><\/span><\/p>\n<p>\u8f49\u9304\u6fc0\u6d3b\u56e0\u5b50\u6a23\u4f5c\u7528\u5b50\uff08transcription activator-like effector, TALE\uff09\u662f\u7531\u67d1\u6a58\u6f70\u760d\u83cc\uff08<em>Xanthomonas<\/em>\uff09\u611f\u67d3\u690d\u7269\u6642\u7b2c\u4e09\u578b\u5206\u6ccc\u7cfb\u7d71\uff08type III secretion system\uff09\u5206\u6ccc\u7684\u86cb\u767d\u8cea\u3002\u6bcf\u500b TALE\u90fd\u5305\u542b\u4e00\u500b\u4e2d\u592e\u91cd\u8907\u5340\u57df\uff0c\u8a72\u5340\u57df\u753134\u500b\u80fa\u57fa\u9178\u7684\u4e0d\u540c\u6578\u91cf\u7684\u91cd\u8907\u55ae\u5143\u7d44\u6210\uff0c\u5176\u4e2d32\u500b\u80fa\u57fa\u9178\u662f\u4fdd\u5b88\u4e14\u7a69\u5b9a\u7684\uff08conserved and stable\uff09\uff0c\u53ea\u6709\u7b2c12\u548c13\u500b\u80fa\u57fa\u9178\u662f\u53ef\u8b8a\u7684\uff08variable\uff09\uff0c\u9019\u5169\u500b\u4f4d\u7f6e-RVDs\uff08repeat variable di-residues\uff09\u5404\u4ee5\u4e0d\u540c\u7684\u983b\u7387\u4f86\u8b58\u5225\u7279\u5b9a\u7684\u6838\u82f7\u9178\uff0c\u4ea6\u53732\u500b\u80fa\u57fa\u9178\u8fa8\u8b581\u500b\u6838\u82f7\u9178\uff0c\u4f8b\u5982\uff1aNI \uff08asparagine-isoleucine\uff09\u8b58\u5225A (adenine, 55\uff05\uff09\uff0cNG\uff08asparagine-glycine\uff09\u8b58\u5225T\uff08thymine, 50\uff05\uff09\uff0cNN\uff08asparagine- asparagine-isoleucine\uff09\u8b58\u5225G\uff08guanine, 7\uff05\uff09\u548cHD\uff08histidine-aspartic acid\uff09\u8b58\u5225C\uff08cytosine, 69\uff05\uff09\u3002\u9650\u5236\u6027\u6838\u9178\u5167\u5207\u9176<em>Fok I<\/em>\u7531N\u7aef\u7279\u7570\u6027DNA\u7d50\u5408\u7d50\u69cb\u57df\u548cC\u7aef\u975e\u7279\u7570\u6027DNA\u5207\u5272\u7d50\u69cb\u57df\u7d44\u6210\uff0c\u5207\u5272\u4f4d\u7f6e\u5728\u8b58\u5225\u4f4d\u4e0b\u6e38\u7b2c9\u548c13\u500b\u6838\u82f7\u9178\u8655\u800c\u5f62\u6210\u96d9\u80a1DNA\u65b7\u88c2\u3002TALEN\uff08transcription activator-like effectors nuclease\uff09\u662f\u4e00\u7a2e\u7531\u5c08\u4e00\u6027\u7684TALE DNA \u7d50\u5408\u7d50\u69cb\u57df\u8207\u975e\u5c08\u4e00\u6027\u7684<em>Fok I<\/em> DNA\u5207\u5272\u7d50\u69cb\u57df\u878d\u5408\u800c\u7522\u751f\u7684\u4eba\u5de5\u9650\u5236\u9175\u7d20\uff08artificial restriction enzyme\uff09\u3002<\/p>\n<p>\u57282021\u5e74\uff0c\u7f8e\u570b\u7814\u7a76\u4eba\u54e1\u5229\u7528\u986f\u5fae\u93e1\u8207\u55ae\u5206\u5b50\u986f\u50cf\u6280\u8853\uff08single-molecule imaging\uff09\u6bd4\u8f03TALEN\u8207CRISPR\u5728\u54fa\u4e73\u52d5\u7269\u7684\u6d3b\u9ad4\u7d30\u80de\u4e2d\u7684\u8868\u73fe\uff0c\u767c\u73feTALEN\u5728\u7dca\u5bc6\u6298\u758a\u7684\u7570\u67d3\u8272\u8cea\uff08heterochromatin\uff09\u5340\u57df\u7684\u8fa8\u8b58\u6548\u7387\u8981\u8f03CRISPR\u9ad8\u51fa5\u500d\uff0c\u800c\u4e14\u57fa\u56e0\u7de8\u8f2f\u6548\u7387\u4ea6\u8f03CRISPR\u70ba\u9ad8\uff0c\u4f46\u662f\u5728\u672a\u6298\u758a\u4e14\u901a\u5e38\u5728\u8f49\u9304\u4e2d\u7684\u771f\u67d3\u8272\u8cea\uff08euchromatin\uff09\u5340\u57df\uff0c\u5247CRISPR\u8868\u73fe\u8f03\u4f73\u3002\u7814\u7a76\u7d50\u679c\u986f\u793a\u82e5\u61c9\u7528\u5728\u7570\u67d3\u8272\u8cea\u4e0a\uff0cTALEN\u662f\u6bd4Cas9\u66f4\u6709\u6548\u7684\u57fa\u56e0\u7de8\u8f2f\u5de5\u5177\u3002\u4ed6\u5011\u63a8\u6e2c\u6703\u51fa\u73fe\u9019\u7a2e\u5dee\u7570\u7684\u539f\u56e0\u53ef\u80fd\u662f\u5169\u7a2e\u57fa\u56e0\u7de8\u8f2f\u6280\u8853\u5c0b\u627e\u6a19\u9776\u4f4d\u9ede\u7684\u65b9\u5f0f\u4e0d\u540c\uff0cTALEN\u540c\u6642\u4f7f\u7528\u5c40\u90e8\u5340\u57df\u641c\u5c0b\uff08local search\uff09\u53ca\u4e09\u5ea6\u7a7a\u9593\u64f4\u6563\uff083D diffusion\uff09\u5169\u7a2e\u65b9\u5f0f\u4f86\u5b9a\u4f4d\u76ee\u6a19\u4f4d\u9ede\uff0c\u4f46\u662fCRISPR \u50c5\u4f9d\u9760\u5c40\u90e8\u5340\u57df\u8490\u5c0b\u3002\u9019\u500b\u7d50\u679c\u5177\u6709\u4e0b\u5217\u91cd\u8981\u610f\u7fa9\uff1a<\/p>\n<p>1.\u4ee5\u5f80\u88ab\u8a8d\u70ba\u96e3\u4ee5\u6539\u9020\u7684\u7570\u67d3\u8272\u8ceaDNA\u5c07\u53ef\u5229\u7528TALEN\u4f86\u9032\u884c\u7de8\u8f2f\uff0c\u6df1\u5177\u6f5b\u529b\u7528\u65bc\u6cbb\u7642\u76f8\u95dc\u75be\u75c5\uff0c\u5982\u6613\u8106\u6027X\u67d3\u8272\u9ad4\u75c7\u5019\u7fa4\uff08fragile-X syndrome\uff09\u53ca\u942e\u72c0\u578b\u7d30\u80de\u8ca7\u8840\u75c7\uff08sickle cell anemia\uff09\u7b49\u3002<\/p>\n<p>2.\u4e0d\u540c\u7684\u57fa\u56e0\u7de8\u8f2f\u6280\u8853\u6839\u64da\u4e0d\u540c\u7684\u539f\u7406\u9032\u884c\u4f5c\u7528\uff0c\u53ef\u4ee5\u9069\u7528\u65bc\u91dd\u5c0d\u4e0d\u540c\u57fa\u56e0\u9ad4\u5340\u57df\u9032\u884c\u7de8\u8f2f\uff0c\u4e14\u5404\u6709\u5176\u64c5\u9577\u4e4b\u8655\uff0c\u53e6\u5916\u53ef\u80fd\u6709\u66f4\u591a\u7684\u57fa\u56e0\u7de8\u8f2f\u5de5\u5177\u5c1a\u5f85\u958b\u767c\uff0c\u80fd\u7528\u65bc\u6539\u5584\u73fe\u6709\u6280\u8853\u7684\u7cbe\u6e96\u5ea6\u53ca\u6548\u7387\u3002<\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: center;\">\u8868\u4e00\uff1aTALEN\u8207CRISPR\u7684\u6bd4\u8f03<\/p>\n<p><img decoding=\"async\" class=\"aligncenter size-full wp-image-36228\" src=\"http:\/\/case.ntu.edu.tw\/blog\/wp-content\/uploads\/2021\/02\/t1-1.png\" alt=\"\" width=\"671\" height=\"521\" srcset=\"https:\/\/case.ntu.edu.tw\/blog\/wp-content\/uploads\/2021\/02\/t1-1.png 671w, https:\/\/case.ntu.edu.tw\/blog\/wp-content\/uploads\/2021\/02\/t1-1-300x233.png 300w\" sizes=\"(max-width: 671px) 100vw, 671px\" 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