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Home > ÀüÁ¦Ç°º¸±â > Genome Editing > ÆíÁý À¯ÀüÀÚ µµÀÔ > AAV CRISPR/SaCas9 Helper Free System (AAV2)
SaCas9¸¦ ÀÌ¿ëÇÑ AAV mediated CRISPR Genome Editing (Single vector)

AAV CRISPR/SaCas9 Helper Free System (AAV2)

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Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Clontech
632619
AAVpro¢ç CRISPR/SaCas9 Helper Free System (AAV2)
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç°  ¶óÀ̼±½º 
1 Kit
(Package)
1,674,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32373, x33227
Clontech
632618
AAVpro¢ç CRISPR/SaCas9 Vector System
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç°  ¶óÀ̼±½º 
1 Kit
(Package)
789,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32373, x33227

¡Ø SpCas9 (from S.pyogenes) ¸¦ ÀÌ¿ëÇÑ AAV meditated CRISPR/Cas9 Genome EditingÀº AAVpro¢ç CRISPR/Cas9 Helper Free System (AAV2) (Code 632608) (Two vector system)À» È®ÀÎÇϼ¼¿ä.
  • Staphylococcus aureus À¯·¡ÀÇ SaCas9¸¦ ÀÌ¿ëÇÑ One vector CRISPR/SaCas9 System
  • Adeno-associated virus (AAV)¸¦ ÀÌ¿ëÇÏ¿© SaCas9°ú sgRNA¸¦ ¼¼Æ÷ ³» µµÀÔ
  • TransfectionÀÌ ¾î·Á¿î Mammlian cell (ºÐ¿­, ºñºÐ¿­¼¼Æ÷)¿¡¼­ Genome Editing
  • AAV¿¡ ÀÇÇÑ Cas9 µµÀÔÀº genome integrationÀ» ¾ïÁ¦ÇÏ°í Cas9ÀÇ ¹ßÇöÀ» Áö¼Ó½Ãų »Ó¸¸ ¾Æ´Ï¶ó, off-target effect¸¦ ¾ïÁ¦
Á¦Ç°¼³¸í
AAVpro¢ç CRISPR/SaCas9 Helper Free SystemÀº single guide RNA (sgRNA)¿Í SaCas9 (from Staphylococcus aureus) À¯ÀüÀÚ¸¦ ¹ßÇöÇÏ´Â Adeno-associated virus 2 (AAV2) particles¸¦ Helper virus ¾øÀÌ Á¦ÀÛ °¡´ÉÇÑ Á¦Ç°ÀÌ´Ù. º» Á¦Ç°Àº S.aureus À¯·¡ÀÇ SaCas9°ú sgRNA¸¦ ÇϳªÀÇ º¤ÅÍ¿¡¼­ ¹ßÇöÇÒ ¼ö ÀÖ´Â single vector systemÀÌ´Ù (±×¸² 1).
S.aureus À¯·¡ÀÇ SaCas9 nucleaseÀÇ À¯ÀüÀÚ Å©±â´Â 3.3 kb·Î½á, ±âÁ¸¿¡ »ç¿ëµÇ¾î¿Ô´ø Streptococcus pyogenes À¯·¡ÀÇ SpCas9 nuclease (4.1 kb) (º¸Åë Cas9À̶ó°í ¸í¸íÇÔ) º¸´Ù ¾à 1 kb Á¤µµ ÀÛ´Ù. ±×·¸±â ¶§¹®¿¡ ÇϳªÀÇ AAV vector¿¡ sgRNA ¹ßÇö À¯ÀüÀÚ¿Í SpCas9 ¹ßÇö À¯ÀüÀÚ¸¦ ÇѲ¨¹ø¿¡ »ðÀÔÀÌ °¡´ÉÇÏ°Ô µÇ¾úÀ¸¸ç, ÀÌ´Â in vitro ¹× in vivo¿¡¼­ ´Ù¾çÇÑ ¼¼Æ÷¿¡¼­ È¿À²ÀûÀ¸·Î Genome editingÀÌ °¡´ÉÇÏ´Ù. ¹Ý¸é, SaCas9ÀÇ °æ¿ì SpCas9°ú PAM ¼­¿­ (Proto-Spacer Adjacent Motif) ¹× target sequenceÀÇ ±æÀÌ (20 - 22 nt)°¡ ´Ù¸£¹Ç·Î sgRNA µðÀÚÀÎ ´Ü°è¸¦ °í·ÁÇÒ ÇÊ¿ä°¡ ÀÖ´Ù (±×¸² 2).
º» Á¦Ç°¿¡ Æ÷ÇԵǾî ÀÖ´Â SaCas9/sgRNA ¹ßÇö AAV vector, AAV packaging¿¡ ÇÊ¿äÇÑ pRC2-mi342 vector ¹× pHelper vector¸¦ HEK293T ¼¼Æ÷ÁÖ¿¡ µµÀÔÀ» Çϸé, Helper virus ¾øÀÌ °í¼øµµ ¹× ³ôÀº TiterÀÇ AAV particleÀ» ¾òÀ» ¼ö ÀÖ´Ù. º» Á¦Ç°¿¡´Â AAV production ¹× CRISPR Genome editingÀ» À§ÇÑ ¸ðµç Á¦Ç° (Ligation mix, Stellar¢â Competent cells, AAV Extraction buffer µî)ÀÌ Æ÷ÇÔ µÇ¾îÀÖ´Ù.


±×¸² 1. pAAV-Guide-it-1 vectorÀÇ SaCas9°ú sgRNA ¹ßÇö
pAAV-Guide-it-1 vector¸¦ ÆÐŰ¡ÇÏ°í ÀÖ´Â AAV particleÀ» Target cell line¿¡ transductionÇÔÀ¸·Î½á, SaCas9 ´Ü¹éÁú°ú sgRNA À¯ÀüÀÚ°¡ µ¿½Ã¿¡ ÇÔ²² ¹ßÇöµÈ´Ù.



±×¸² 2. SaCas9ÀÇ PAM sequence
SaCas9ÀÇ PAM ¼­¿­Àº NNGRR (N=A, T, G, C / R=A/T)À̸ç, PAMÀ¸·ÎºÎÅÍ 3 - 4 bpÀÇ ¿°±â¸¦ Àý´ÜÇÑ´Ù. ¶ÇÇÑ, sgRNAÀÇ Target sequence´Â 20 - 22nt¸¦ ±ÇÀåÇÑ´Ù. SaCas9ÀÇ sgRNA µðÀÚÀÎÀº http://benchling.com¿¡¼­ °¡´ÉÇÏ´Ù.



±×¸² 3. AAV¸¦ ÀÌ¿ëÇÑ SaCas9°ú SpCas9ÀÇ Genome editing È¿À² ºñ±³
CYP2 Exon1 À¯ÀüÀÚ Áß, 2°³ÀÇ ´Ù¸¥ ¹è¿­À» editing Ç¥ÀûÀ¸·Î ÇÏ¿© Two vector systemÀÎ AAVpro¢ç CRISPR/Cas9 Helper Free System (AAV2) (Code 632608) °ú Sinlge vector systemÀÎ AAVpro¢ç CRISPR/SaCas9 Helper Free System (AAV2) (Code 632619)¸¦ »ç¿ëÇÏ¿© AAV mediated CRISRP/Cas9 editingÀ» ¼öÇàÇÏ¿´´Ù. 12-well plate¿¡ 1 x 105°³ÀÇ ¼¼Æ÷¸¦ seeding, over night ¹è¾ç ÈÄ, SpCas9 or SaCas9 virus vector¸¦ 1 x 105 MOI·Î °¨¿°½ÃÄ×´Ù. 72½Ã°£ ÈÄ, ¼¼Æ÷¸¦ ȸ¼öÇÏ°í Genome DNA º¯À̵µÀÔ È¿À²À» Guide-it¢â Mutation Detection Kit (Code 631443)À» »ç¿ëÇÏ¿© ºÐ¼® ¹× ºñ±³ÇÏ¿´´Ù.
Applications
AAV-based delivery of a user-defined sgRNA and Cas9 for mammalian genome editing using CRISPR/Cas9 technology
±¸¼ºÇ° (ÀÚ¼¼ÇÑ ³»¿ëÀº CoA¸¦ ÂüÁ¶Çϼ¼¿ä)
AAVpro¢ç CRISPR/SaCas9 Helper Free System (AAV2) (Cat. No. 632619)
  • AAVpro¢ç CRISPR/SaCas9 Vector Set
  • Guide-it¢â Ligation Components
  • Stellar¢â Competent Cells
  • pRC2-mi342 Vector
  • pHelper Vector
  • AAVpro¢ç Extraction Solution

AAVpro¢ç CRISPR/SaCas9 Vector System (Cat. No. 632618)
  • AAVpro¢ç CRISPR/SaCas9 Vector Set
  • Guide-it¢â Ligation Components
  • Stellar¢â Competent Cells
º¸Á¸
Stellar¢â Competent Cells: -80 ¡É
±âŸ: -20 ¡É
(AAVpro¢ç Extraction SolutionÀº À¶ÇØ ÈÄ, ½Ç¿Â º¸°ü)




Keyword : AAV,AAV CRISPR,in vivo,µµÀÔÈ¿À²,saCas9,CRISPR,CRISPR/Cas9,Cas9,sgRNA,Å©¸®½ºÆÛ,gene editing,À¯ÀüÀÚÆíÁý

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