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Genome Editing
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ÆíÁý À¯ÀüÀÚ µµÀÔ
> CRISPR Plasmid System
Single vector¸¦ ÀÌ¿ëÇÑ È¿À²ÀûÀÎ sgRNA, Cas9 ¹ßÇö
CRISPR Plasmid System
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Clontech
632601
Guide-it¢â CRISPR/Cas9 System (Green)
1 System
(Package)
728,000¿ø
,
Clontech
632602
Guide-it¢â CRISPR/Cas9 System (Red)
1 System
(Package)
728,000¿ø
,
Product index
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..
Single vector system:
ÇϳªÀÇ º¤ÅÍ¿¡¼ target-specific sgRNA, Cas9 nuclease, Çü±¤´Ü¹éÁú (ZsGreen1 ¶Ç´Â tdTomato) ¹ßÇö
Exceptionally bright fluorescent protein:
trasnfection È¿À²À» È®ÀÎÇÒ ¼ö ÀÖ´Â ¸®Æ÷Åͷνá EGFPº¸´Ù 2.5¹è~6¹è ´õ ¹àÀº ZsGreen1
¶Ç´Â tdTomato »ç¿ë
Simple Plasmid construction:
vector°¡ ¹Ì¸® ¼±ÇüÈ(Pre-linearized vector)µÇ¾îÀÖ¾î ¿øÇÏ´Â À¯ÀüÀÚÀÇ ÇÕ¼º sgRNA¸¦ °£´ÜÈ÷
ligation & clone
Complete system:
sgRNA/Cas9 system¿¡ ÇÊ¿äÇÑ Á¦Ç°ÀÌ ¸ðµÎ Æ÷ÇÔ
Á¦Ç°¼³¸í
Guide-it CRISPR/Cas9 system
Á¦Ç°Àº CRISPR/Cas9 ±â¼úÀÇ mammalian genome modification (or genome editing)À» À§ÇØ target single guide RNAs(sgRNAs)ÀÇ Å¬·Î´× ¹× ¹ßÇöÀ» ÇÒ ¼ö ÀÖµµ·Ï Á¦ÀÛµÈ Á¦Ç°ÀÌ´Ù. º» Á¦Ç°¿¡ Æ÷ÇԵǾîÀÖ´Â º¤ÅÍ´Â Cas9 nuclease, target-specific sgRNA, Çü±¤´Ü¹éÁúÀ» ¹ßÇöÇÒ ¼ö ÀÖÀ¸¸ç, ¹ßÇöµÈ Çü±¤´Ü¹éÁúÀº transfection È¿À²À» ¸ð´ÏÅ͸µ Çϰųª Flow cytometry¸¦ ÀÌ¿ëÇÏ¿© transfected cellÀ» isolating/enriching ¿¡ »ç¿ëÇÒ ¼ö ÀÖ´Ù.
±×¸² 1. The structure of the pGuide-it vector.
This vector is part of the Guide-it CRISPR/Cas9 Systems, kits for the cloning of a target single guide RNA (sgRNA) for CRISPR/Cas9 genome editing. The pGuide-it vector is used to simultaneously express Cas9 nuclease, a target-specific sgRNA, and an exceptionally bright fluorescent protein (either ZsGreen1 or tdTomato). Cas9 and the fluorescent protein are co-expressed from a bidirectional CMV promoter, and a user-defined sgRNA is cloned downstream of a human U6 promoter. To construct the vector, a pair of oligos corresponding to the genomic target (your guide sequence) are annealed to form a duplex. Then, the duplexed DNA is cloned into the pre-linearized vector using the included high-efficiency ligation mix and competent cells.
±×¸² 2. Genomic DNA modifications introduced using the pGuide-it-ZsGreen1 plasmid.
1 x 10
5
cells (HEK 293, HeLa, U2OS, or HT1080 cell lines) were seeded in 12-well plates 16 hr prior to transfection. Cells were transfected with 2.5 ¥ìg of pGuide-it-ZsGreen1 plasmid using the
Xfect Transfection Reagent
(Code 631317); the plasmid expressed Cas9, ZsGreen1, and an sgRNA targeting the either CCR5, C4BPB, or EMX1. After 48 hr, fluorescence was analyzed by microscopy (A), and by FACS (B, Transfection efficiency). The efficiency of gene modification was also evaluated using the
Guide-it Mutation Detection Kit
(Code 631443), a PCR-based method for testing for the presence of indels (B). The target sequence was amplified directly from cells, the amplicon was melted and hybridized to form mismatched targets that were cleaved with Guide-it Resolvase. Cleavage products were resolved on an agarose gel and quantified by densitometry to estimate the frequency of indels (B, Indel).
Àû¿ë
Cloning and expression of a target single guide RNA (sgRNA) for mammalian genome editing using CRISPR/Cas9 technology
±¸¼ºÇ° (ÀÚ¼¼ÇÑ ³»¿ëÀº CoA¸¦ ÂüÁ¶Çϼ¼¿ä)
Guide-it CRISPR/Cas9 System (Green) (Cat. No. 632601)
pGuide-it-ZsGreen1 Vector (Linear)
Guide-it Ligation Components
Stellar Competent Cells
Guide-it CRISPR/Cas9 System (Red) (Cat. No. 632602)
pGuide-it-tdTomato Vector (Linear)
Guide-it Ligation Components
Stellar Competent Cells
º¸Á¸
Stellar Competent Cells:-70¡É
±âŸ:-20¡É
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Keyword : CRISPR vector,Cas9 vector,CRISPR,CRISPR/Cas9,Cas9,sgRNA,Å©¸®½ºÆÛ,gene editing,À¯ÀüÀÚÆíÁý
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Cas9 Ç×ü (Poly/Mono)
mRNA, sgRNA, siRNAµî ´Ù¾çÇÑ RNA transfection..
RNA Transfection Reagent
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