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Àΰ£À» Æ÷ÇÔÇÑ ´Ù¾çÇÑ »ý¸íüÀÇ °Ô³ð¼¿ÀÌ ºü¸£°Ô ¹àÇôÁö°í ÀÖÀ½¿¡ µû¶ó ¼¿ÀÇ ±â´ÉÀ» ºÐ¼®ÇÏ´Â °Í¸¸ÀÌ ¾Æ´Ñ Á÷Á¢ÀûÀ¸·Î editingÇÏ·Á´Â ¿¬±¸µéÀÌ ÁøÇàµÇ°í ÀÖ´Ù. °Ô³ðÀ» editingÇÏ´Â ¹æ¹ý Áß Áö±Ý±îÁö ´ëÇ¥ÀûÀ¸·Î ¸¹ÀÌ ¾²ÀÌ°í ÀÖ´Â ±â¼ú·Î´Â ZFN(Zinc finger nuclease), TALEN(Transcription Activator-Like Effector Nucleases)ÀÌ ÀÖ´Ù. ÀÌ µÎ °¡Áö´Â ´Ü¹éÁúÀ» ÀÌ¿ëÇÏ¿© °Ô³ð»óÀÇ Æ¯Á¤ DNA ¼¿À» ÀνÄÇÏ°í ±× ºÎºÐÀ» À߶󳻴 ±â¼ú·Î½á ƯÁ¤ ¼¿¿¡ mutationÀ» ÀÏÀ¸Å°°Å³ª homologous recombinationÀ» ÀÌ¿ëÇÏ¿© ¿øÇÏ´Â ¼¿À» °Ô³ð¿¡ insertionÇÏ´Â ÇüÅ·Π¾²¿©¿Ô´Ù. µÎ °¡Áö ±â¼ú ¸ðµÎ °Ô³ð»óÀÇ specificÇÑ site¸¦ targetÇÒ ¼ö ÀÖ´Ù´Â Å« ÀåÁ¡À» °¡Áö°í ÀÖÀ¸³ª µÎ ´Ü¹éÁú ¸ðµÎ Å©±â°¡ Ä¿¼ ÇÕ¼ºÇϴµ¥ µ·°ú ½Ã°£ÀÌ ¸¹ÀÌ µç´Ù´Â ´ÜÁ¡À» °¡Áö°í ÀÖ´Ù.
ZFN°ú TALENÀ» ´ëüÇÒ ¼ö ÀÖ´Â ±â¼ú·Î½á RNA¸¦ ÀÌ¿ëÇÏ¿© °Ô³ðÀ» editingÇÏ´Â ±â¼úÀÎ CRISPR (Clustered, Regularly Interspaced, Short Palindromic Repeats) – Cas(CRISPR associated protein) systemÀÌ ÃÖ±Ù Á¦½ÃµÇ¾ú´Ù. CRISPR-Cas systemÀº º»·¡ archaea¿Í bacteriaÀÇ adaptive immunity systemÀ¸·Î ¾Ë·ÁÁ® ÀÖ´Ù. Virus³ª plasmid°¡ ħÀÔÇÏ¿´À» ¶§ archaea³ª bacteria´Â crRNA¿Í tracrRNA¶ó°í ºÒ¸®´Â ªÀº RNA¸¦ ¸¸µé¾î ³»°í ÀÌ RNA´Â ħÅõÇÑ DNA¿¡ °áÇÕÇÑ´Ù. ÀÌ ±¸Á¶¸¦ Cas ´Ü¹éÁúÀÌ ÀνÄÇÏ°í DNA¸¦ À߶ó ºÐÇؽÃÅ°¸ç ÀÚ½ÅÀ» ¿ÜºÎÀÇ DNA ħÀÔÀ¸·ÎºÎÅÍ º¸È£ÇÑ´Ù. ƯÁ¤ ¼¿À» ÀνÄÇÏ¿© ±× ºÎºÐÀ» À߶󳻴 systemÀÌ ZFN°ú TALEN°ú À¯»çÇÏ¿© À̸¦ ÀÀ¿ëÇÏ¿© °Ô³ð editing¿¡ »ç¿ëÇÏ·Á´Â ¿¬±¸µéÀÌ È°¹ßÈ÷ ÁøÇàµÇ¾ú´Ù.
Á¾ ¸¶´Ù ´Ù¾çÇÏ°Ô Á¸ÀçÇÏ´Â CRISPR-Cas system¿¡´Â Å©°Ô ¼¼°¡Áö ŸÀÔÀÌ Á¸ÀçÇÑ´Ù. ÀÌ Áß Cas9 ´Ü¹éÁú°ú crRNA, tracrRNA°¡ °ü¿©ÇÏ´Â typeII°¡ ´ëÇ¥ÀûÀÌ¸ç °¡Àå Àß ¾Ë·ÁÁ® ÀÖ´Ù. crRNA¿Í tracrRNAÀÇ ±¸Á¶¿Í À¯»çÇÑ ÇüŸ¦ °¡Áö´Â single guide RNA¸¦ ÇÕ¼ºÇÑ µÚ Cas9 ´Ü¹éÁú°ú ÇÔ²² ÀÌ¿ëÇÏ¿© ¿©·¯ organismÀÇ genomeÀ» editingÇÏ·Á´Â ¿¬±¸µéÀÌ ÁøÇàµÇ°í ÀÖ´Ù. (Kira S Makarova et al. (2011))
2013³âµµ nature¿Í cell¿¡ CRISPR-Cas systemÀ» ÀÌ¿ëÇÏ¿© mamalian cellÀÇ °Ô³ð editing¿¡ ¼º°øÇÑ »ç·ÊµéÀÌ º¸°íµÇ¾ú´Ù. Cas9 ´Ü¹éÁúÀ» vectorÇüÅ·Πhuman cell³»¿¡¼ Á÷Á¢ ¹ßÇö½ÃÅ°°í, single guide RNA¸¦ in vitro¿¡¼ ÇÕ¼ºÇÏ¿© human cell¿¡ ÁÖÀÔ½ÃŲ °á°ú target site¿¡ mutationÀ» ¸¸µé ¼ö ÀÖ´Ù´Â °ÍÀÌ È®ÀεǾúÀ¸¸ç ±âÁ¸ÀÇ ZFNÀ̳ª TALEN°ú´Â ´Ù¸£°Ô off target effect°¡ ¾øÀ½ÀÌ Áõ¸íµÇ¾ú´Ù. (Seung Woo Cho et al. (2013)) ¶ÇÇÑ Cas9 ´Ü¹éÁú°ú synthetic single guide RNA¸¦ ¸ðµÎ human cell³»¿¡ vectorÀÇ ÇüÅ·Π³Ö°í ¹ßÇö½ÃÄÑ °Ô³ðÀ» editingÇÑ °á°úµµ º¸°íµÇ¾ú´Ù. (Prashant Mali et al. (2013))
Human cell »Ó ¾Æ´Ï¶ó CRISPR-Cas system type II¸¦ ÀÌ¿ëÇÏ¿© zebrafish embryo genome »óÀÇ 11°³ target site¿¡ mutationÀ» ÀÏÀ¸Å³ ¼ö ÀÖ´Ù´Â °ÍÀÌ È®ÀεǾú´Ù. Cas9 ´Ü¹éÁúÀ» mRNAÇüÅ·ΠÇÕ¼ºÇÑ µÚ single guide RNA¿Í ÇÔ²² microinjectionÀ» ÅëÇØ cell ³»·Î ÁÖÀÔ½ÃÄÑ CRISPR-Cas systemÀ» ±¸ÇöÇÑ °á°ú ±âÁ¸ ZFNÀ̳ª TALENÀ¸·Î´Â editing ÇÒ ¼ö ¾ø¾ú´ø ºÎºÐÀÇ editingÀÌ °¡´ÉÇÔÀÌ º¸°íµÇ¾ú´Ù. (Woong Y Hwang et al. (2013)) ±âÁ¸ÀÇ ZFN°ú TALENó·³ ÇÑ °÷ÀÇ target site¸¦ Á¤ÇÏ¿© mutationÀ» ÀÏÀ¸Å² °Í ¿Ü¿¡ µÎ Á¾·ùÀÇ single guide RNA¸¦ ÀÌ¿ëÇÏ¿© CRISPR-Cas systemÀ¸·Î multiplex genome engineeringÀ» °¡´ÉÇÏ°Ô ÇÑ ±â¼úµµ °³¹ßµÇ¾ú´Ù. (Le cong et al. (2013))
GenomeÀ» editingÇÒ ¶§ ZFNÀ̳ª TALEN°ú °°Àº °æ¿ì¿£ ´Ù¸¥ site¸¦ detectionÇÏ·Á¸é »õ·Î¿î proteinÀ» designÇØ¾ß Çß´Ù. ÇÏÁö¸¸ CRISPR-Cas systemÀ» ÀÌ¿ëÇÒ °æ¿ì °°Àº protein (Cas9)À» »ç¿ëÇÏ¸é¼ targetÇÏ´Â sequence¿¡ µû¶ó crRNAÀÇ sequence¸¸ ¹Ù²ãÁÖ¸é µÇ±â ¶§¹®¿¡ ZFNÀ̳ª TALEN¿¡ ºñÇØ °¡°ÝÀûÀ̳ª ½Ã°£ÀûÀÎ ¸é¿¡¼ ¸¹Àº ´ÜÃàÀ» ÇÒ ¼ö ÀÖ´Ù. ÇÏÁö¸¸ ¾ÆÁ÷±îÁö ÀÌ systemÀº ¸î°¡Áö ´ÜÁ¡À» °¡Áö°í ÀÖ´Ù. ù°·Î Cas9 proteinÀÌ ÀνÄÇÏ´Â PAM sequence(NGG)°¡ target genome site¿¡ Á¸ÀçÇØ¾ß ÇÑ´Ù´Â ÇÑ°èÁ¡ÀÌ Á¸ÀçÇÑ´Ù. ÇÏÁö¸¸ ÀÌ´Â Cas9 ´Ü¹éÁú engineeringÀ¸·Î ÇØ¼ÒµÉ ¼ö ÀÖ´Â ÇÑ°èÁ¡À̶ó »ý°¢µÈ´Ù. ¶ÇÇÑ chromatinÀÇ ±¸Á¶¿Í epigenetic state°¡ eukaryotic cellÀÇ °Ô³ð editing¿¡ ¿µÇâÀ» ÁÙ ¼ö ÀÖ´Â ¿ä¼Ò·Î Áö¸ñ ¹Þ°í ÀÖ´Ù. ÀÌ ºÐ¾ß¿¡ ´ëÇÑ ¿¬±¸°¡ ´õ ÀÌ·ç¾î Áø´Ù¸é CRISPR-Cas systemÀº °Ô³ðÀÇ ¸ðµç ƯÁ¤ site¸¦ ½±°í ºü¸£°Ô ½ÉÁö¾î multiplex engineeringÀ¸·Î±îÁö »ç¿ë µÉ ¼ö ÀÖ´Â °¡´É¼ºÀ» °¡Áö°í ÀÖ´Ù°í º¸¿©Áø´Ù.
- 2011, Kira S Makarova et al. Evolution and classification of the CRISPR-Cas system Nature review June 2011
volume9 467- 477
- 2013 Seung Woo Cho et al. Nature Biotechnology 29 January 2013; doi:10.1038/nbt.2507 Targeted genome
engineering in human cells with the Cas9 RNA-guided endonuclease
- Prashant Mali et al. Science vol339 15 February 2013 RNA-guided human genome engineering via Cas9
- 2013 Woong Y Hwang et al. Nature Biotechnology 29 January 2013; doi:10.1038/nbt.2501 Efficient genome
editing in zebrafish using a CRISPR-Cas system
- Le cong et al. Science vol339 15 February 2013 Multiplex genome engineering using CRISPR/Cas systems
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