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Genome Editing
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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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Clontech
632619
AAVpro¢ç CRISPR/SaCas9 Helper Free System (AAV2)
1 Kit
(Package)
1,674,000¿ø
,
Clontech
632618
AAVpro¢ç CRISPR/SaCas9 Vector System
1 Kit
(Package)
789,000¿ø
,
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Overivew
CRISPR/Cas9 Overview
CRISPR ½ÇÇè °¡À̵å (Tu..
About Guide-it¢â Kits
CRISPR Selection Guide
[Cancer Research] Gene..
Customer data for Guid..
Guide-it¢â À¯ÀüÀÚ ÆíÁý..
Knock-in FAQ
À¯ÀüÀÚ ÆíÁý Àü ´Ü°è
sgRNA ÇÕ¼º°ú ½ºÅ©¸®´×
[Upgrade] Guide-it¢â L..
Genome-Wide sgRNA Libr..
ÆíÁý À¯ÀüÀÚ µµÀÔ
CRISPR Plasmid System
CRISPR/Cas9 Gesicle Pr..
AAV CRISPR/Cas9 Vector..
AAV CRISPR/SaCas9 Help..
Lenti-X¢â CRISPR/Cas9 ..
Cas9 Ç×ü (Poly/Mono)
RNA Transfection Reage..
iPSC Single-Cell Cloni..
Recombinant Cas9 ´Ü¹é..
À¯ÀüÀÚ ÆíÁý ÈÄ È®ÀÎ
Mutation Detection Kit
¿°±âġȯ, »ðÀÔ º¯ÀÌ °Ë..
Genotype Confirmation ..
Indel Identification K..
CRE recombinase
CRE Recombinase Gesicl..
AAV Helper Free System..
¡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 10
5
°³ÀÇ ¼¼Æ÷¸¦ seeding, over night ¹è¾ç ÈÄ, SpCas9 or SaCas9 virus vector¸¦ 1 x 10
5
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
ÆíÁý À¯ÀüÀÚ µµÀÔ
Single vector¸¦ ÀÌ¿ëÇÑ È¿À²ÀûÀÎ sgRNA, Cas9 ..
CRISPR Plasmid System
Off-target effect °¨¼Ò, Hard-to-transfect ce..
CRISPR/Cas9 Gesicle Production System
Adeno-associated virus delivery-CRISRP/Cas9 ..
AAV CRISPR/Cas9 Vector System
SaCas9¸¦ ÀÌ¿ëÇÑ AAV mediated CRISPR Genome E..
AAV CRISPR/SaCas9 Helper Free System (AAV2)
Lentivirus¸¦ ÀÌ¿ëÇÑ sgRNA, Cas9 gene deliver..
Lenti-X¢â CRISPR/Cas9 System
Cas9 nuclease °ËÃâÀ» À§ÇÑ Ç×ü
Cas9 Ç×ü (Poly/Mono)
mRNA, sgRNA, siRNAµî ´Ù¾çÇÑ RNA transfection..
RNA Transfection Reagent
hiPS cellÀÇ single cell ¹è¾ç, À¯ÀüÀÚ ÆíÁý¿¡ ..
iPSC Single-Cell Cloning DEF-CS¢â Culture Media Kit
Electroporation delivery¸¦ À§ÇÑ Cas9 ´Ü¹éÁú
Recombinant Cas9 ´Ü¹éÁú