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       Genome engineeringÀ» ÅëÇÑ Áö´ÉÇü Àΰø¹Ì»ý¹° °³¹ß
       sklee@unist.ac.kr        
       À̼º±¹        2013.11.05 17:55        19489
´Ù¿î·Îµå : Genome engineeringÀ» ÅëÇÑ Áö´ÉÇü Àΰø¹Ì»ý¹° °³¹ß(UNIST À̼º±¹ ±³¼ö).pdf(1 M)
 

Genome engineeringÀ» ÅëÇÑ Áö´ÉÇü Àΰø¹Ì»ý¹° °³¹ß

 

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 ÃÖ±Ù ¸î ³â°£ ¹Ì±¹ÀÇ CNN1 , ¿µ±¹ÀÇ BBC2 ¿Í ±¹¿µ¹æ¼Û3 µî ¿©·¯ ±ÇÀ§ ÀÖ´Â ¹Ìµð¾î¸Åü¸¦ ÅëÇØ ÇÕ¼º»ý¹°ÇÐÀÇ ÀáÀ缺ÀÌ º¸µµµÇ¾ú´Ù. ÇöÀç °¡Àå ¶ß°Å¿î °ü½ÉÀÌ ¸ð¾ÆÁö°í ÀÖ´Â ºÐ¾ßÀÎ ÇÕ¼º»ý¹°ÇÐÀº ±âÁ¸ ¸î °³ÀÇ À¯ÀüÀÚ¸¦ ´ë»óÀ¸·Î ÇÏ´Â À¯Àü°øÇÐÀû ½ÇÇè ¹æ¹ýÀ̳ª ±â¼úµé¿¡ ±¹ÇÑµÈ °ÍÀÌ ¾Æ´Ñ, ƯÁ¤ À¯ÀüüÀÇ Àü¹ÝÀûÀÎ ÀÌÇظ¦ ¹ÙÅÁÀ¸·Î ÇÑ´Ù. ƯÈ÷ »õ·Î¿î »ýü´ë»ç°æ·ÎÀÇ Á¦ÀÛ»Ó ¾Æ´Ï¶ó ³ª¾Æ°¡ À¯Àüü ¼³°è ¹× Àΰø À¯Àüü ÇÕ¼º µîÀ» °¡´ÉÇÏ°Ô ÇÏ¿© Áö±Ý±îÁö ´Ù·ç¾îÁø ÀûÀÌ ¾ø´Â »õ·Î¿î ºÐ¾ß¸¦ ´Ù·ç´Â Çй®À¸·Î ¿©°ÜÁö°í ÀÖ´Ù.


 Áö³­ ¹Ý¼¼±â µ¿¾È »ý¹°ÇкоßÀÇ ÇÑ Áٱ⿡ ºÒ°úÇß´ø À¯Àü°øÇÐÀº ±Þ°ÝÇÑ ¼ºÀåÀ» ¹ÙÅÁÀ¸·Î »ý¹°Ã¼¸¦ ±â¹ÝÇÑ ¸ñÀû ´ë»ç»ê¹°ÀÇ »ý»ê·®À» »ó½Â½ÃÅ°°Å³ª ´Ù¸¥ »ý¹°Ã¼B·ÎºÎÅÍ »ý¹°Ã¼A¿¡´Â ¾ø´Â ¹°ÁúÀ» »ý»êÇÏ°Ô ÇÏ´Â µîÀÇ ÀϵéÀ» ½ÇÇö °¡´ÉÇÏ°Ô ÇÏ¿´´Ù. Áö³­ °íÀüÀû »ý¹°ÇÐÀû ¹æ¹ýÀ» ÅëÇÑ »ý¹°Ã¼ÀÇ À¯ÀüÀÚ º¯ÇüÀº ¸î °¡Áö°¡ ÀÖ¾î ¿ÔÀ¸³ª, ÃʱâÀÇ ¿¬±¸ÀÚµéÀº ¹°¸®•È­ÇÐÀûÀÎ ¹æ¹ýÀ» »ç¿ëÇÏ¿© À¯ÀüÀÚ¿¡ µ¹¿¬º¯À̸¦ µµÀÔÇÏ¿´´Ù.4 5 ºñ·Ï À¯ÀüÀÚ¿¡ µ¹¿¬º¯À̸¦ µµÀÔ½ÃÄÑ »õ·Î¿î À¯ÀüÀÚÇü(genotype)Àº ¾ò¾úÁö¸¸ ¿øÇϴ ǥÇöÇü(phenotype)À» Á¦ÀÛÇϱâ´Â ½±Áö ¾ÊÀº ÀÏÀ̾ú´Ù. BiomoleculeÀÇ »ý»ê ¹× ºÐÇØ ±×¸®°í ±×µéÀÇ »óÈ£ÀÛ¿ëÀ» ÀÌÇØÇϱâ À§ÇÑ ¹æ´ëÇÑ ¾çÀÇ Áö½ÄÀÇ °á¿©¿Í ÇÔ²² ¸ðµ¨¸µÀ» À§ÇÑ computational sourceÀÇ ¾àÇÑ ±â¹ÝÀº »ý¸íüÀÇ ÀÌÇظ¦ ¹ÙÅÁÀ¸·Î °øÇÐÀûÀÎ °³³äÀ» µµÀÔÇϴ°íÀÚ ÇÏ´Â ÇÕ¼º»ý¹°ÇÐÀÇ ¹ßÀüÀÇ Å« Àå¾Ö¹°À̾ú´Ù. ÇÏÁö¸¸ ¹«¾ùº¸´Ùµµ À¯ÀüÀÚ ¼­¿­ÀÇ º¯È­¸¦ À§ÇÑ ºÐÀÚ»ý¹°ÇÐÀûÀÎ ±â¼úÀÇ ºÎÀç´Â ¿øÇϴ ǥÇöÇüÀ» ¾ò±â À§ÇØ °èȹÀû º¯°æ(rational alteration)º¸´Ù ÁøÈ­Àû ÃÖÀûÈ­(evolutionary optimization)¿¡ ÀÇÁ¸Çϵµ·Ï ÇÏ¿´´Ù. ÇÏÁö¸¸ Áö³­ ¸î ³â°£ ¼öõ Á¾ÀÇ »ý¸íü À¯ÀüÀÚ ¼­¿­ºÐ¼®À» ÅëÇؼ­ »ý¹° Á¤º¸·®ÀÌ ±âÇϱ޼öÀûÀ¸·Î Áõ°¡ÇÏ°í ±×¿Í µ¿½Ã¿¡ °è»êÀû »ý¹°ÇÐÀû µµ±¸(computational tool)ÀÇ ¹ßÀüÀ¸·Î »ý¸í½Ã½ºÅÛÀ» ÀÌÇØÇÏ°Ô µÇ°í À̸¦ ¹ÙÅÁÀ¸·Î »õ·Î¿î »ý¸í ½Ã½ºÅÛ ¹× »ý¸íü¸¦ Á¦ÀÛÇÏ°íÀÚ ÇÏ´Â ÇÕ¼º»ý¹°ÇÐÀÌ ±Þ¼ÓÀûÀ¸·Î ¹ßÀüÇÏ°Ô µÈ´Ù. ½ÇÁúÀûÀ¸·Î Àΰø »ý¸í ½Ã½ºÅÛÀ» Á¦ÀÛÇϱâ À§Çؼ­´Â ´Ù¾çÇÑ Áö³ð¿£Áö´Ï¾î¸µ(genome engineering) ±â¼úÀÇ µµ¿òÀÌ ²À ÇÊ¿äÇÏ´Ù.


 ÃÖ±Ù °³¹ßµÇ°í ÀÖ´Â Áö³ð¿£Áö´Ï¾î¸µ ±â¹ýµéÀº ÇÕ¸®ÀûÀÎ À¯ÀüüÀÇ ÀÌÇظ¦ ¹ÙÅÁÀ¸·Î ÇÑ À¯ÀüÀÚ ÁöµµÀÇ ¼³°è¿Í ÇÕ¼ºÀ» ÅëÇØ ´ë»ç»ê¹°ÀÇ »ý»ê·®À» Áõ°¡½Ãų ¼ö ÀÖ´Â Àü·«ÀÓ°ú µ¿½Ã¿¡ ´Ù¾çÇÑ Áö´ÉÇü Àΰø »ý¸íü¸¦ Á¦ÀÛÇÒ ¼ö ÀÖ´Â ±â¹ÝÀ» ¸¸µé°í ÀÖ´Ù. ÀÌ¿¡ ÃÖ±Ù¿¡ °³¹ßµÈ Àΰø ¹Ì»ý¹° Á¦ÀÛ ±â¼úÀÎ Áö³ð¿£Áö´Ï¾î¸µ ±â¼úµé°ú ÇÕ¼ºµÈ »õ·Î¿î »ý¹°Ã¼µé Áß ¿ì·®±ÕÁÖÀÇ ¼±º°À» È¿°úÀûÀ¸·Î µµ¿ï ¼ö ÀÖ´Â bioMEMS ±â¼ú¿¡ ´ëÇؼ­ °£·«È÷ ¼³¸íÇÏ°íÀÚ ÇÑ´Ù.


 °¡. Áö³ð ¿£Áö´Ï¾î¸µ ±â¼úµé

 

   1. Transcriptional activator-like effector genome editing

 

±×¸² 1. Transcriptional activator-like effector nuclease¸¦ ÀÌ¿ëÇÑ genome engineering  (http://en.wikipedia.org/wiki/Tal_effector_nuclease)

 

 Áö³ð¿£Áö´Ï¾î¸µÀÌ À¯ÀüÀÚ ¼­¿­ÀÇ Æ¯Á¤ºÎºÐÀ» ÀνÄÇÏ°í Ç¥ÀûÇÏ´Â °ÍÀ» ÀüÁ¦·Î Çϴ Ư¼º´öºÐ¿¡ zinc-finger(ZF)³ª transcriptionl activator-like(TAL) effector¿Í °°Àº DNA-°áÇÕ ´Ü¹éÁúÀÌ ÇϳªÀÇ Áö³ð¿£Áö´Ï¾î¸µ ±â¼úÀÌ µÉ ¼ö ÀÖ¾ú´Ù. Á¦ÇÑÈ¿¼Ò¿Í °°Àº DNA º¯ÇüÈ¿¼Ò¿Í ¿¬°áµÈ DNA-°áÇÕ ´Ü¹éÁúÀº ƯÁ¤ DNA¼­¿­¿¡ °áÇÕÇÑ ÈÄ À߶óÁÜÀ¸·Î½á ÀÌÁß°¡´ÚÀý´Ü(double strand break; DSB)¸¦ Çü¼º ÇÒ ¼ö ÀÖ°í »óµ¿Àû ÀçÁ¶ÇÕ(homologous recombination)À̳ª ºñ»óµ¿ ¸»´Ü ºÀÇÕ(non-homologous end joining; NHEJ)À» ÅëÇÏ¿© mutationÀ» µµÀÔÇÒ ¼ö ÀÖ¾ú´Ù. Ãʱ⿡ °¢±¤À» ¹Þ¾Ò´ø ZF-nuclease´Â motif´ç ¾à 3°³ÀÇ base pairs (bp)¸¦ ÀνÄÇÒ ¼ö ÀÖ¾úÁö¸¸ motif¸¦ stackingÇÏ´õ¶óµµ »ó´ëÀûÀ¸·Î ³·Àº ƯÀ̼º°ú ½ÇÇè½Ç¼öÁØ¿¡¼­ ¾ò±â¿£ ºñ½Ñ ´ÜÁ¡À¸·Î ÀÎÇØ ÃÖ±Ù¿¡´Â TALEN(TAL effector nuclease)¸¦ ÀÌ¿ëÇÏ¿© Áö³ð¿£Áö´Ï¾î¸µÀ» ÇÏ´Â Ãß¼¼ÀÌ´Ù. ºñ·Ï ÇÑ motif´ç ÇϳªÀÇ bp¸¦ ÀνÄÇÏ°í Å« size·Î ÀÎÇØ assembly°¡ º¹ÀâÇÑ ÆíÀÌÁö¸¸ ±ä DNA¼­¿­À» ÀνÄÇϱâ À§ÇÑ stackingÀÌ ºñ±³Àû ½±±â ¶§¹®¿¡ Áö³ð¿£Áö´Ï¾î¸µ ±â¼ú·Î ¸¹ÀÌ »ç¿ëµÇ°í ÀÖ´Ù. Iowa state universityÀÇ Yang`s ÆÀÀº ÃÖ±Ù TALEN¸¦ ÀÌ¿ëÇÏ¿© È¿¸ð(Saccharomyces cerevisiae)ÀÇ À¯Àüü¿¡ ÀÖ´Â 3°³ÀÇ À¯ÀüÀÚÁ¦°Å(gene knockout)¿¡ ¼º°øÇÏ¿´´Ù.6 23bp±îÁö Àνİ¡´ÉÇÑ TALENÀ» ÀÎÀ§ÀûÀ¸·Î Á¦ÀÛÇÏ¿© È¿¸ðÀÇ Æ¯Á¤ À¯ÀüÀÚ¸¦ Á¦°ÅÇÏ¿´°í ¿¹»óµÇ¾îÁö´Â Ç¥ÇöÇüÀ» ¾òÀ» ¼ö ÀÖ¾ú´Ù. ¶ÇÇÑ »óµ¿Àû ÀçÁ¶ÇÕÀ» ÅëÇÑ À¯ÀüÀÚ ±³Ã¼µµ 34%ÀÇ È¿À²À» º¸¿´´Ù. ÀÌ·¯ÇÑ TALEN È°¿ë Áö³ð¿£Áö´Ï¾î¸µÀº Àúºñ¿ë°ú ³ôÀº È¿À²·Î ¿øÇϴ ǥÇöÇüÀ» ¾òÀ» ¼ö ÀÖ´Â »õ·Î¿î ±â¹ýÀ¸·Î ÁøÇÙ »ý¹°ÀÇ À¯ÀüÀÚ º¯Çü¿¡ »ó´çÇÑ ¿ªÇÒÀ» ÇÒ °ÍÀ¸·Î ±â´ëµÇ¾îÁø´Ù.7 8

 

   2. Genome editing using an engineered type II CRISPR system

 

 

±×¸² 2. (a,b) Mechanism of CRISPR system. (Philippe H et al, Science, 2010) (c,d) Human genome editing with    

           engineered type II CRISPR system. (Mali P et al, Science, 2013.)

 

 ¿øÇÙ»ý¹°ÀÇ ÀûÀÀ¸é¿ª½Ã½ºÅÛ ¿ª½Ã ÁÁÀº Áö³ð¿£Áö´Ï¾î¸µ µµ±¸°¡ µÉ ¼ö ÀÖ´Ù. ÆÄÁö°¨¿°(phage infection)À̳ª ¼¼Æ÷°£ Á¢ÇÕ(conjugation)¿¡ ÀÇÇØ ¼¼Æ÷³»·Î À¯ÀÔµÈ ¿Ü·¡ À¯ÀüÀÚ¿Í °áÇÕ ÇÒ ¼ö ÀÖ´Â RNA¿Í Cas9 nuclease°¡ º¹ÇÕü¸¦ ÀÌ·ï DSB¸¦ ¾ß±âÇÏ´Â CRISPR(clustered regularly interspaced short palindromic repeats)ÀÇ ¿ø¸®¸¦ ±âº»À¸·Î ÇÏ´Â »õ·Î¿î ¹æ¹ýÀÌ ¹Ù·Î ±× ¿¹ÀÌ´Ù. ÃÖ±Ù ÇϹöµå ¸ÞµðÄà ½ºÄðÀÇ Church`s ÆÀÀº CRISPRÀÇ ½Ã½ºÅÛÀ» ÀÌ¿ëÇÏ¿© Æ÷À¯µ¿¹°¼¼Æ÷(mammalian cell)¿¡¼­ non-sense GFP¸¦ activeÇÑ ÇüÅ·Π¹Ù²Ù¾ú´Ù.9 Cas9 nuclease¿Í °áÇÕÇÒ ¼ö ÀÖ´Â gRNA(guide RNA)¿Í Ÿ°Ù À¯ÀüÀÚ¸¦ ÀνÄÇÏ´Â RNA¸¦ °áÇÕ½ÃÄÑ ¼¼Æ÷³»·Î »ðÀÔ½ÃÅ°°í ±×¿Í µ¿½Ã¿¡ ÄÚµ· ÃÖÀûÈ­µÈ Cas9À» Á¦ÀÛÇÏ¿© ¼¼Æ÷³»·Î µµÀÔ½ÃÅ°°Ô µÇ¸é RNA°¡ Ÿ°Ù DNA¼­¿­°ú RNA-DNA base pairingÀ» Çü¼ºÇÏ°í Cas9ÀÇ nucleaseÈ°¼ºÀÌ DSB¸¦ Çü¼ºÇÏ°Ô µÈ´Ù. ±×·Î ÀÎÇØ »óµ¿Àû ÀçÁ¶ÇÕÀÌ ÃËÁøµÇ°Ô µÇ¹Ç·Î ¿øÇÏ´Â À¯ÀüÀÚÇüÀ» µµÀÔÇÒ ¼ö ÀÖ°Ô µÈ´Ù. ´õ¿í ½Å±âÇÑ °ÍÀº Cas9 nuclease°¡ ¹ßÇöµÉ ¶§¿Í ±×·¸Áö ¾ÊÀ» ¶§ÀÇ »óµ¿Àû ÀçÁ¶ÇÕ È®·üÀÌ °ÅÀÇ 8¹è³ª Â÷ÀÌ°¡ ³­´Ù´Â °ÍÀÌ´Ù. ÀÌ´Â Áö¼ÓÀûÀÎ Cas9 nucleaseÀÇ ¹ßÇöÀÌ »óµ¿Àû ÀçÁ¶ÇÕÀ» ÃËÁøÇÑ´Ù´Â °ÍÀ» ¾Ï½ÃÇϱ⵵ ÇÑ´Ù. ÀÌ·¯ÇÑ CRISPR ½Ã½ºÅÛÀº ³·°Ô´Â 2%¿¡¼­ ³ô°Ô´Â 25%±îÁö À¯ÀüÀÚ º¯Çü È¿À²À» º¸¿´´Ù. ÀÌ °á°ú´Â RNA-guided editingÀº ºü¸£°í multiplexibleÇÑ Áö³ð¿£Áö´Ï¾î¸µ µµ±¸·Î½á °¡´É¼ºÀ» º¸¿©Áá°í ÀÌ¿¡ µû¶ó Ãß°¡ÀûÀÎ ¿¬±¸µéÀÌ ÁøÇàµÇ°í ÀÖ´Â Ãß¼¼ÀÌ´Ù.10

 

   3. Trackable multiplex recombineering(TRMR) method

±×¸² 3. (a) Trackable multiplex recombineering(TRMR) method (b) Multiple strategy to rapidly generate cell mixtures with

           defined genetic modification. (Warner JR et al, Nat Biotechnol, 2010)

 

 ¹Ì»ý¹° Áö³ðÀ» ÀÌÇØÇϱâ À§Çؼ­´Â ¸¹Àº ¼öÀÇ library°¡ ÇÊ¿äÇÏ´Ù´Â °ÍÀ» °¨¾ÈÇÑ´Ù¸é ÀûÀýÇÑ À¯ÀüÀÚº¯ÀÌ(genetic variation)À» Á¦ÀÛÇÑ ÈÄ¿¡ ±×·ÎºÎÅÍ ÆÄ»ýµÇ´Â ¿¬±¸¸¦ ÇÏ´Â °ÍÀÌ ½Ã°£ÀûÀ¸·Î³ª ºñ¿ëÀûÀ¸·Î³ª ÈξÀ °æÁ¦ÀûÀ̶ó´Â °ÍÀº ´©±¸µµ ºÎÁ¤ÇÏÁö ¸øÇÒ °ÍÀÌ´Ù. ÃÖ±ÙÀÇ Áö³ðÇÐ(genomics)ÀÇ ¹ß´Þ°ú multiplex DNA ÇÕ¼º ±â¼ú, ±×¸®°í »óµ¿Àû ÀçÁ¶ÇÕ ±â¼úÀÇ ¹ß´ÞÀº ÀÌ·¯ÇÑ ÀϵéÀÌ °¡´ÉÇÏ°Ô ÇÏ°í ÀÖ´Ù. Áö³ðÇÐÀÇ ¹ß´ÞÀº À¯ÀüÀÚ Á¦°Å¿Í °°Àº ¹æ¹ýÀ» ÅëÇÏ¿© À¯ÀüÀÚÀÇ Æ¯¼ºÀ» ÆľÇÇϴµ¥ µµ¿òÀ» ÁÖ¾ú°í ÀÌ´Â À¯ÀüÀÚÀÇ ¿ªÈ°À» È®ÀÎÇÒ ¼ö ÀÖ´Â °­·ÂÇÑ µµ±¸°¡ µÇ¾úÁö¸¸ ´Ù¼ö À¯ÀüÀÚµéÀÇ µ¹¿¬º¯ÀÌ¿¡ ÀÇÇØ ¾ß±âµÇ´Â º¹ÇÕÀûÀÎ º¯È­¿¡¼­ Ç¥ÇöÇüÀ» ¿¹ÃøÇÏ°í ºÐ¼®ÇÏ´Â °Í¿¡´Â ¾ÆÁ÷ ¾Æ½¬¿î ¼º°ú¸¦ º¸ÀÌ°í ÀÖ¾ú´Ù. ÃÖ±Ù °³¹ßµÈ TRMRÀº ±âÁ¸ÀÇ ¹®Á¦Á¡À» º¸¿ÏÇÏ°í ½±°í ºü¸£°Ô ´Ù¼öÀÇ µ¹¿¬º¯À̸¦ Á¦ÀÛÇÒ ¼ö ÀÖ´Â ¹æ¹ýÀ¸·Î ¼Ò°³µÇ¾ú´Ù. ´Ü 1Àϸ¸¿¡ Àüü ´ëÀå±ÕÀÇ À¯ÀüÀÚÀÇ 95%¸¦ º¯ÇüÇÏ¿´°í À̷κÎÅÍ ¿øÇϴ ǥÇöÇüÀÇ ¹Ì»ý¹°À» ¼±º°ÇÒ ¼ö ÀÖ¾ú´Ù.11 ±âÁ¸¿¡ ÈçÈ÷ »ç¿ëµÇ´ø on-off¹æ½ÄÀÌ ¾Æ´Ñ up-down¹æ½ÄÀ¸·Î À¯ÀüÀÚÀÇ ¹ßÇöÀ» Á¶ÀýÇÔÀ¸·Î½á Ÿ°Ù À¯ÀüÀÚ¿¡ ´ëÇØ º¸´Ù Æø ³ÐÀº ÀÌÇظ¦ Á¦°øÇÏ¿´´Ù. ÀÎÀ§ÀûÀ¸·Î ÇÕ¼ºµÈ dsDNA´Â rolling circle amplification¿¡ ÀÇÇØ ÁõÆøµÇ°í ¼¼Æ÷³»¿¡¼­ Áö³ðÀ¸·Î »ðÀÔµÊÀ¸·Î½á Ÿ°Ù À¯ÀüÀÚÀÇ ¹ßÇöÀ» Á¶ÀýÇÏ°Ô µÇ´Âµ¥ À̶§ ¸¸µé¾îÁö´Â mutant library´Â ¼±º°°úÁ¤À» °ÅÄ¡¸é¼­ ¿øÇϴ ǥÇöÇüÀÇ ¼¼Æ÷¸¸À» ¼±º°ÇÒ ¼ö ÀÖ´Ù. ÀÌ·¯ÇÑ °úÁ¤À» ÅëÇØ ´Ü ÀÏÁÖÀϸ¸¿¡ ¿øÇÏ´Â ÇüÁú¿¡ ¿µÇâÀ» ÁÖ´Â À¯ÀüÀÚµéÀ» È®ÀÎÇÒ ¼ö ÀÖ¾ú´Ù. ´ë´Ù¼öÀÇ À¯ÀüÀÚµéÀº »õ·ÎÀÌ ¹àÇôÁø À¯ÀüÀÚµéÀ̾úÀ¸¸ç ÀϺδ ±âÁ¸¿¡ ÀÌ¹Ì ¹àÇôÁ® ÀÖ´ø À¯ÀüÀÚ¿´°í ±× Áß¿¡¼­´Â »õ·Î¿î ±â´ÉÀÌ ¹àÇôÁö±âµµ ÇÏ¿´´Ù. TRMRÀ» ÅëÇؼ­ ¹«¼öÈ÷ ¸¹Àº mutant library¸¦ Á¦ÀÛÇÒ ¼ö ÀÖÀ¸¸ç À̸¦ ÅëÇØ switches, oscillators ȤÀº sensors¿Í °°Àº Ãß°¡ÀûÀÎ ¿ªÇÒÀ» ÇÏ´Â À¯ÀüÀڵ鿡 ´ëÇؼ­µµ ±Ô¸íÇÒ ¼ö ÀÖÀ» °ÍÀÌ´Ù.12

 

    4. Multiplex automated genome engineering

±×¸² 4. Rapid and continuous generation of sequence diversity by MAGE (Wang HH et al, Nature, 2009)

 

 ¸¹Àº Áö³ð¿£Áö´Ï¾î¸µ ±â¼úÀÌ ÇϳªÀÇ À¯ÀüÀÚ µ¹¿¬º¯ÀÌ¿¡ ÁÖ·Î ÃÊÁ¡À» ¸ÂÃá µ¥¿¡´Â ÀÌÀ¯°¡ ÀÖ´Ù. ZFNÀ̳ª TALEN, ±×¸®°í group II intron systemÀ» ÀÌ¿ëÇÑ ±â¹ýÀº ¸ðµÎ DSB¸¦ ¾ß±âÇϱ⠶§¹®¿¡ ´Ù¼öÀÇ À¯ÀüÀÚÀÇ µ¹¿¬º¯ÀÌ¿¡´Â ÀûÀýÄ¡ ¾Ê´Ù. ÀÌ·¯ÇÑ DSB°¡ ´Ù¼ö »ý¼ºµÇ°Ô µÈ´Ù¸é Àüü Áö³ðÀ» Æı«ÇÒ »Ó ¾Æ´Ï¶ó NHEJ¿¡ ÀÇÇØ ÀǵµÄ¡ ¾ÊÀº rearrangement¸¦ ¹ß»ý½ÃŲ´Ù. ¶ÇÇÑ ¥ë-red system¿¡ ÀÇÇØ ¸Å°³µÇ´Â dsDNA ÀçÁ¶ÇÕ ¿ª½Ã ³·Àº È¿À²À» º¸À̱⠶§¹®¿¡ ´Ù¼öÀÇ µ¹¿¬º¯ÀÌ Á¦ÀÛ¿¡´Â ÀûÀýÄ¡ ¾Ê´Ù. ÀÌ·¯ÇÑ ¹®Á¦Á¡À» º¸¿ÏÇϱâ À§Çؼ­ ÃÖ±Ù¿¡´Â dsDNA°¡ ¾Æ´Ñ ssDNA¸¦ ÀÌ¿ëÇÏ´Â ¹æ¹ýÀÌ ¼Ò°³µÇ¾ú´Ù. MAGE (multiplex automated genome engineering)Àº ÀÛÀº ssDNA°¡ lagging strandÀÇ Okazaki fragment ÇÕ¼º¿¡ ÇÁ¶ó¸Ó(primer)·Î ÀÛ¿ëÇÏ¿© µþ¼¼Æ÷ Áö³ð¿¡ µµÀԵǸ鼭 À¯ÀüÀÚ º¯À̸¦ À¯µµÇÑ´Ù.13 MAGE´Â ssDNA¸¦ ¼¼Æ÷³»·Î µµÀÔ½ÃÅ´À¸·Î½á ¾ÆÁÖ °£´ÜÇÏ°Ô ´Ù¼öÀÇ µ¹¿¬º¯À̸¦ Á¦ÀÛÇÒ ¼ö ÀÖ¾ú´Ù. ¾à 30~100bpÀÇ ssDNA´Â »óµ¿ÀÇ ¸»´Ü¸¸ ÀÖÀ¸¸é Ÿ°Ù À¯ÀüÀÚ ºÎÀ§¿¡ »óº¸ÀûÀ¸·Î °áÇÕÇÔÀ¸·Î½á ¿ì¸®°¡ ¿øÇÏ´Â À¯ÀüÀÚ ¼­¿­À» µþ¼¼Æ÷·Î µµÀÔ½Ãų¼ö ÀÖ´Ù.14 ºñ½ÁÇÑ ¿ø¸®·Î MAGE´Â mismatch repair¿¡ °ü¿©ÇÏ´Â mutS À¯ÀüÀÚ¸¦ Á¦°Å½ÃÅ´°ú µ¿½Ã¿¡ ¥ë-red systemÀÇ ¹ßÇöÀ» ÅëÇØ ssDNA »ðÀÔÀ» 1000¹è±îÁöµµ Áõ°¡½Ãų¼ö ÀÖ¾ú´Ù. MAGE¿Í °°ÀÌ ssDNA-mediated recombinationÀÇ °æ¿ì ´Ù¸¥ plasmid³ª dsDNAº¸´Ù À¯ÀüÀÚ µµÀÔ(transformation)È®·üÀÌ ³ô±â ¶§¹®¿¡ º¸´Ù ³ôÀº È¿À²ÀÇ À¯ÀüÀÚ ÀçÁ¶ÇÕÀ» ±â´ëÇÒ ¼ö ÀÖÀ» »Ó¸¸ ¾Æ´Ï¶ó ÇÑ cycle´ç ¼Ò¿äµÇ´Â ½Ã°£ÀÌ »ó´ëÀûÀ¸·Î ´Ù¸¥ ±â¹ýµéº¸´Ù ª¾Æ ¸¹Àº ¼öÀÇ µ¹¿¬º¯ÀÌ ±ÕÁÖ¸¦ ¸¸µé ¼ö ÀÖ´Ù. ¶ÇÇÑ °£´ÜÇÑ ¿ø¸®´öºÐ¿¡ ´Ù¾çÇÑ ±ÕÁÖ¿¡¼­µµ Àû¿ë°¡´ÉÇÒ °ÍÀ¸·Î ¿¹»óµÈ´Ù. ssDNA-mediated recombinationÀÇ ¸¹Àº ÀåÁ¡ ´öºÐ¿¡ ÇöÀçµµ ¸¹Àº ¿¬±¸µéÀÌ Ãß°¡ÀûÀ¸·Î ÁøÇàµÇ°í ÀÖ´Â »óȲÀÌ´Ù.15 16 17 18

 

 ³ª. ¹Ì¼¼À¯Ã¼ÀåÄ¡¸¦ ÀÌ¿ëÇÑ ¿ì¼ö ±ÕÁÖ ¼±º°

 

 

±×¸² 5. ´Ù¾çÇÑ ¹Ì¼¼À¯Ã¼ÀåÄ¡°¡ ÀÀ¿ëµÈ »ç·Êµé

 

 ´Ù¾çÇÑ ÇÕ¼º»ý¹°ÇÐÀû ±â¼úÀ» »ç¿ëÇؼ­ À籸¼ºµÈ »õ·Î¿î À¯Àüü´Â °¢°¢ÀÇ °³Ã¼µéÀÌ ¸ðµÎ ´Ù¸¥ È°¼ºÀ» °¡Áö°Ô ÇÑ´Ù. ¿¹¸¦ µé¾î À§¿¡ ¼³¸íµÈ MAGE¿Í °°Àº ¹æ¹ýÀ¸·Î À籸¼ºµÈ DNA¸¦ °¡Áø ±ÕÁÖµéÀÌ ¸¸µé¾îÁö¸é, À̸¦·ÎºÎÅÍ ¿øÇϴ ǥÇöÇüÀÇ ±ÕÁÖ¸¦ ¼±ÅÃÀûÀ¸·Î ºÐ¸®Çϱâ À§Çؼ­´Â °íÀüÀû ¹æ¹ýÀ¸·Î´Â ¸¹Àº ³ëµ¿·Â°ú ½Ã°£ÀÌ ÇÊ¿äÇß¾ú´Ù. ÇÏÁö¸¸ ÃÖ±Ù ¹Ì¼¼À¯Ã¼°øÇÐ(microfluidics)µî°ú °°Àº »õ·Î¿î ±â¼úÀÌ µîÀåÇϸ鼭 Á» ´õ ¼Õ½±°í, ºü¸£°í È¿°úÀûÀ¸·Î °í¼Ó´ë·®½ºÅ©¸®´× (high-throughput screening; HTS)À» °¡´ÉÇÏ°Ô ÇÏ¿´´Ù. ÇÕ¼º»ý¹°Çаú´Â ¿ÏÀüÈ÷ ´Ù¸¥ ¿¬±¸ºÐ¾ßÀÎ ¹Ì¼¼À¯Ã¼°øÇÐÀÌ ÇÔ²² Çù¾÷(collaboration)µÇ¾î ¼º°øÀûÀϼö ÀÖ´Ù´Â °¡´É¼ºÀ» º¸¿©ÁÖ¾ú´ø »ç·Ê´Â 2005³â ½ºÅÄÆ÷µåÀÇ ÄùÀÌÅ© ¿¬±¸±×·ìÀÌ ÀÚµ¿È­µÈ ¹Ì¼¼À¯Ã¼ÀåÄ¡ ³»¿¡¼­ ¹Ì»ý¹°ÀÇ »ýÀåÀ» ÃËÁø, ÀúÇØ, À¯µµ µî°ú °°ÀÌ ´Ù¾çÇÑ ¹æ¹ýÀ» ÅëÇØ ¹«·Á 40ÀÏ°£ °üÂûÇß´ø °ÍÀÌ Àß ¾Ë·ÁÁ® ÀÖ´Ù.19 ¶ÇÇÑ, CITÀÇ Leadbetter ±×·ìÀº microfluidic ÀåÄ¡¸¦ ÀÌ¿ëÇØ ±âÁ¸¿¡ À¯ÀüÁ¤º¸°¡ ÀüÇô ¾Ë·ÁÁ®ÀÖÁö ¾Ê¾Ò´ø ¹Ì»ý¹° TraponemaÀÇ ´ë·® 106°³ÀÇ PCRÀ» Çس´Ù. ÀÌ´Â bioMEMs ±â¼úÀÇ ÀåÁ¡ÀÎ ´ë·®ÀÇ sample 󸮰¡´É´É·Â°ú ¹ÝÀÀ¿¡ ±Ø¼Ò·®ÀÇ chemical¸¸ÀÌ ÇÊ¿äÇÑ Á¡À» ½ÊºÐ»ì·Á, target DNA ÀÚ¼¼ÇÑ Á¤º¸¸¦ ¾ËÁö ¸øÇصµ ƯÁ¤ geneÀÇ Á¸Àç¿©ºÎ¸¦ °¡·Á³¾ ¼ö ÀÖ´Â PCRµîÀÇ ±â¼ú¿¡ Àû¿ë ÇÒ ¼ö ÀÖ´Ù´Â °ÍÀ» º¸¿©ÁØ´Ù.20


 À§¿¡ ¼³¸íµÈ µÎ°¡Áö ¹Ì¼¼À¯Ã¼ ÀåÄ¡¸¦ ±âÁ¡À¸·Î ÇÑ ´Ù¾çÇÑ Áö³ð¿£Áö´Ï¾î¸µ±â¼úÀº ¹Ì¼¼À¯Ã¼ÀåÄ¡¸¦ ÀÌ¿ëÇÑ ±â¼úµé°ú ÇÔ²² ÃÖ±Ù±îÁö Àû¿ëµÇ¾îÁ® ¿Ô´Ù. ¿¹¸¦ µé¸é, °¡Àå ±âº»ÀûÀÎ PCR°ú °°Àº ±â¼úµéÀÌ ¹Ì¼¼À¯Ã¼ÀåÄ¡³»¿¡¼­ ÀçÇöµÇ¾ú°í21. À§¿¡ ¼³¸íµÈ ÄùÀÌÅ© ±×·ìÀÇ bioreactor´Â ¿©ÀüÈ÷ ¸¹Àº °ü½ÉÀ» ¹Þ°í ÀÖÀ¸¸ç, DNA sequence22, ƯÁ¤µÈ ´Ü¹éÁúÀÇ °Ë·®, °ËÃâÀÌ °¡´ÉÇÑ ÀÚµ¿È­µÈ ÀåÄ¡23, Å©±â³ª ´Ù¸¥ ¿ÜºÎ ¿äÀÎÀ» »ç¿ëÇÑ »ý¹°Ã¼ÀÇ separationÀ§ÇÑ ¹Ì¼¼À¯Ã¼ÀåÄ¡24, ´Ü½Ã°£³»¿¡ 107~9°³ÀÇ »ùÇà ºÐ¼®ÀÌ °¡´ÉÇÑ ±¸È¹È­µÈ ¹Ì¼¼¹æ¿ï »ý¼º ¹× ÀúÀåÀåÄ¡25 µîµî ¿©·¯°¡Áö ¹æ¹ýµéÀÌ ÇÕ¼º»ý¹°ÇÐ ºÐ¾ß ¿¬±¸¿¡ È°¿ëµÇ°í ÀÖ´Ù.

 

±×¸² 6. High-throughput screening of strains (Jina Y et al, Nat commun, 2013)

 

 ÀÌ·¯ÇÑ Áö³ð¿£Áö´Ï¾î¸µÀ» Á¢¸ñÇÑ ÇÕ¼º»ý¹°Çаú ´ë»ç°øÇÐÀº ±²ÀåÈ÷ ¸¹Àº platform ±ÕÁÖµéÀ» ¸¸µé¾î³Â°í ethanol26, higher alcohol27, fatty acids28 µîÀÌ ¸¹Àº ÀçÁ¶ÇÕ ±ÕÁÖµé·ÎºÎÅÍ »ý»êµÇ°í ÀÖ´Ù. ÃÖ±Ù¿¡´Â MAGE¿Í TRMR°ú °°ÀÌ ´ÙÁß¿£Áö´Ï¾î¸µ ±â¼úÀÌ ¹ß´ÞÇÔ¿¡ µû¶ó ¼ö¸¹Àº ¹Ì»ý¹°±ºÁý¿¡¼­ ÀûÀýÇÑ ±ÕÁÖ¸¦ ¼±º°Çϱâ À§ÇÑ ½ºÅ©¸®´× ±â¼úÀÌ ÇÊ¿äÇÏ°Ô µÇ¾ú´Ù. ÇÏÁö¸¸ Çü±¤¹°ÁúÀ» ÅëÇÑ Æ¯Á¤ ±ÕÁÖ ¼±º°29À̳ª enrichment¿Í °°Àº ¼±ÅþÐ(selective pressure)À» »ç¿ëÇÏ´Â ±âÁ¸ÀÇ HTS30´Â ¸¹Àº ½Ã°£ÀÌ ¼Ò¸ðµÈ´Ù´Â ´ÜÁ¡À» °¡Áö°í ÀÖ´Ù. ÀÌ¿¡ µû¶ó »ý¹°ÇÐÀû ½ºÅ©¸®´×º¸´Ù Á» ´õ ½±°í ºü¸¥ ¹æ¹ýÀ¸·Î bioMEMs°¡ ¶°¿À¸£°í ÀÖ°í °ü·Ã ¿¬±¸°¡ ¼öÇàµÇ°í ÀÖ´Ù. ÀÌó·³ Áö³ð ¿£Áö´Ï¾î¸µÀº ÇÕ¼º»ý¹°ÇÐ ¹× ´ë»ç°øÇÐÀÇ ¹ßÀüÀ» À§ÇÑ Ãæ½ÇÇÑ µµ±¸·Î½á ±×¸®°í bioMEMs´Â »õ·Î¿î ÆÄÆ®³Ê·Î½á Àΰø¹Ì»ý¹°ÀÇ °³¹ß¿¡ Áß¿äÇÑ ¿ªÇÒÀ» ÇÏ°í ÀÖ´Ù.


 

Âü°í¹®Çå

 

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