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Electroporation delivery¸¦ À§ÇÑ Cas9 ´Ü¹éÁú

Recombinant Cas9 ´Ü¹éÁú

-

Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Clontech
632640
Guide-it¢â Recombinant Cas9 (Electroporation-Ready)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
3x100 ug (632641 x 3)
903,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32692
Clontech
632641
Guide-it¢â Recombinant Cas9 (Electroporation-Ready)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
100 ug
334,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â
Clontech
632678
Guide-it¢â Recombinant Cas9 (10 µg/µl)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
200 ug
795,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x254749
Clontech
632679
Guide-it¢â Recombinant Cas9 (10 µg/µl)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
500 ug
1,480,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â





Á¦Ç° Ư¡

Code

Á¦Ç°¸í

´Ü¹éÁú ³óµµ

Glycerol ³óµµ

#632641, #632640

Guide-it¢â Recombinant Cas9 (Electroporation-Ready)

3 ¥ìg/¥ìl

10% ¹Ì¸¸

#632678, #632679

Guide-it¢â Recombinant Cas9 (10 ¥ìg/¥ìl)

10 ¥ìg/¥ìl

50%


  • The Cas9 protein solution is sterile and well-tolerated by mammalian cells
  • Highly efficient editing when combined with Guide-it Complete sgRNA Screening System (Code 632636)
  • Minimizes off-target effects-better than plasmid-based delivery systems
  • Works well in hard-to-transfect cell lines
Á¦Ç°¼³¸í
Guide-it¢â Recombinant Cas9 (Electroporation-Ready)Àº Streptococcus pyogenes À¯·¡ÀÇ Cas9 ÀçÁ¶ÇÕ ´Ü¹éÁú·Î½á CRISPR/Cas9 gene editing ½ÇÇè¿¡ »ç¿ëÇÒ ¼ö ÀÖµµ·Ï C-terminal nuclear-localization signal (NLS) º¯ÇüÀÌ µÇ¾î ÀÖ´Ù. Cas9-sgRNA ribonucleoprotein(RNP) º¹ÇÕüÀÇ electroporationÀº ºü¸¥ ½Ã°£ ³» gene editingÀÌ °¡´ÉÇϵµ·Ï Çϸç, ¶ÇÇÑ off-target effect¸¦ ÃÖ¼ÒÈ­ ÇÒ ¼ö ÀÖ´Ù.
º» Á¦Ç°Àº Transfection ½Ã¾à¿¡ ÀÇÇÑ RNP delivery¿¡µµ Àû¿ë °¡´ÉÇϸç, ÀÌ °æ¿ì MirusÀÇ TransIT-X2 Dynamic Delivery System (Code MIR 6000) »ç¿ëÀ» ÃßõÇÑ´Ù (±×¸² 4. ÂüÁ¶)

±×¸² 1. Gene knockouts in the Cellartis hiPSC-18 cell line. The strength of the cleaved bands gives a semiquantitive estimate of the percentage of edited cells. As can be seen, the Guide-it recombinant Cas9, when combined with sgRNA from the Guide-it In Vitro Transcription Kit, provides consistent and effective gene editing for many targets. Numbers at the bottom of each gel represent gene editing efficiencies (expressed as a %) for the indicated RNPs.


±×¸² 2. Homology-directed repair at the AAVS1 or CXCR4 genes. A template containing a HindIII restriction site was inserted into the AAVS1 gene, and a template containing both a HindIII and a BamHI restrictrion site was inserted into the CXCR4 gene. Gene targets were then amplified from target cells using PCR and analyzed by restriction digest. Editing efficiencies are shown above each positive well on the gel. The extra band in the BamHI digest of the CXCR4 gene is due to a second BamHI site already present in the wild-type gene before editing.


±×¸² 3. Guide-it¢â Recombinant Cas9 (Electroporation-Ready)°ú Ÿ»ç Cas9 ´Ü¹éÁúÀÇ Konck-out È¿À² ºñ±³
Guide-it¢â Recombinant Cas9 (Electroporation-Ready)°ú Ÿ»çÀÇ Cas9 proteinÀ» ÀÌ¿ëÇÏ¿©, Human iPS cell¿¡¼­ CD81 À¯ÀüÀÚ¸¦ Ÿ°ÙÇÏ´Â sgRNA-Cas9 RNP º¹ÇÕü¸¦ Neon Transfection System (Thermo Fisher Scientific)À¸·Î Electroporation µµÀÔÇÏ¿´´Ù. ±× °á°ú, Ÿ»çÀÇ Cas9 ´Ü¹éÁú¿¡ ºñÇÏ¿© ¿ùµîÈ÷ ³ôÀº Knock-out È¿À²À» ¾òÀ» ¼ö ÀÖ¾ú´Ù (ÀÚ»ç ½ÇÇèµ¥ÀÌÅÍ, n=2x3 times)


±×¸² 4. Guide-it¢â Recombinant Cas9 (Electroporation-Ready)¿Í TransIT-X2¸¦ ÀÌ¿ëÇÑ iPS cell gene editing
PPIB (Cyclophili B)¿¡ ´ëÇÑ 2°³ÀÇ guide RNA (gRNA)¿Í Cas9 protein (Ÿ»ç Á¦Ç° ¶Ç´Â Guide-it Recombinant Cas9 protein) º¹ÇÕü (RNP)¸¦ Çü¼ºÇÏ¿´´Ù. ±× ÈÄ, RNP º¹ÇÕü¸¦ TransIT-X2 Dynamic Delivery System (Code MIR 6000) (1§¡/well ÷°¡, 24-well plate, Mirus Bio) ¸¦ ÀÌ¿ëÇÏ¿© iPS cell (KOLF-2, Wellcome Trust Sanger Institute)¿¡ Transfection ¹æ½ÄÀ¸·Î µµÀÔÇÏ¿´´Ù. ¿©·¯ ³óµµÀÇ guide RNA¸¦ Á¶ÇÕÇÑ RNP º¹ÇÕü µµÀÔ ÈÄ, T7E1 mismatch detectionÀ¸·Î Àý´Ü È¿À²À» È®ÀÎÇÑ °á°ú Ÿ»ç Á¦Ç° ´ëºñÇÏ¿© º¸´Ù ³ôÀº Àý´ÜÈ¿À²À» È®ÀÎÇÒ ¼ö ÀÖ¾ú´Ù.
°ü·Ã Á¦Ç°
Guide-it Complete sgRNA Screening System (Code 632636)
- Gene delivery¿¡ Àû¿ë°¡´ÉÇÑ 10-20ugÀÇ °í¼öÀ², °í¼øµµ sgRNA ÇÕ¼º
- sgRNA candidates Áß °¡Àå editing È¿À²ÀÌ ³ôÀº sgRNA ½ºÅ©¸®´×

TransIT-X2 Dynamic Delivery System (Code MIR 6000)
- DNA, siRNA, miRNA °¢°¢ ¶Ç´Â Co-transfection °¡´ÉÇÑ ¸¸´É Transfection ½Ã¾à
- CRISPR/Cas9À» À§ÇÑ sgRNA-Cas9 RNP delivery¿¡µµ È¿À²Àû
Application Note
TransIT-X2 Dynamic Delivery System (Code MIR 6000)À» ÀÌ¿ëÇÑ Genome Editing ¿¹½Ã
- 293T Cell Line Àû¿ë ¿¹½Ã (Ŭ¸¯)
- K562 Cell Line Àû¿ë ¿¹½Ã (Ŭ¸¯)
- Human iPS Cell Àû¿ë ¿¹½Ã (Ŭ¸¯)




Keyword : RNP,Ribonucleoprotein,Cas9 protein,Knockout,Knock out,KO,Knock-out,KI,Knock in, Knockin,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