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Home > ÀüÁ¦Ç°º¸±â > Genome Editing > ÆíÁý À¯ÀüÀÚ µµÀÔ > CRISPR/Cas9 Gesicle Production System
Off-target effect °¨¼Ò, Hard-to-transfect cell lineÀ» À§ÇÑ CRISPR/Cas9

CRISPR/Cas9 Gesicle Production System

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Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Clontech
632613
Guide-it¢â CRISPR/Cas9 Gesicle Production System
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç°  ¶óÀ̼±½º 
1 Kit
(Package)
1,434,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32688, x33374
Clontech
632616
Guide-it¢â CRISPR/Cas9 Gesicle Packaging Set
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
1 Set
965,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32688
Clontech
632617
Gesicle Producer 293T Cell Line
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç°  ¶óÀ̼±½º 
1 ml
737,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32676, x32688
Clontech
632612
pGuide-it-sgRNA1 Vector System
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç°  ¶óÀ̼±½º 
1 Kit
(Package)
692,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32688, x33374

Guide-it CRISPR/Cas9 Gesicle ½ÇÇè ¿ø¸® ¹× °³¿ä (Ŭ¸¯)

  • Cell-derived nanovesicle(Gesicle)À» ÀÌ¿ëÇÏ¿© Cas9/sgRNA ribonucleoprotein(RNP)¸¦ ¼¼Æ÷ ³» Á÷Á¢ µµÀÔ
  • Broad tropism: ´Ù¾çÇÑ Cell line¿¡ Àû¿ë °¡´É
  • Hard-to-transfection cell line (ºÐ¿­¼¼Æ÷, ºñºÐ¿­¼¼Æ÷, iPS ¼¼Æ÷)¿¡¼­µµ È¿À²ÀûÀÎ CRISPR/Cas9 Genome Editing °¡´É
  • Off-target effect ÃÖ¼ÒÈ­: µµÀÔµÈ Cas9/sgRNA Complex´Â ¼¼Æ÷ ³»¿¡¼­ ½Å¼ÓÇÏ°Ô Á¦°ÅµÇ¾î Off-target effect¸¦ ÁÙÀÏ ¼ö ÀÖÀ½
Á¦Ç°¼³¸í
Guide-it CRISPR/Cas9 Gesicle Production systemÀº Cas9/sgRNA Complex¸¦ ¼¼Æ÷¿¡ È¿À²ÀûÀ¸·Î µµÀÔÇϱâ À§ÇÑ Cas9/sgRNA GesicleÀ» Á¦ÀÛÇÏ´Â Á¦Ç°ÀÌ´Ù. Cas9/sgRNA GesicleÀ» ÀÌ¿ëÇϸé Plasmid transfectionÀ̳ª Virus transductionÀ» ÀÌ¿ëÇÑ Genome Editing°ú ºñ±³ÇÏ¿© ´õ¿í ´Ù¾çÇÑ ¼¼Æ÷ÁÖ¿¡ È¿À²ÀûÀ¸·Î Cas9/sgRNA ribonucleoprotein (RNP)¸¦ µµÀÔÇÒ ¼ö ÀÖ´Ù.
º» ½Ã½ºÅÛ¿¡¼­´Â target DNA specific-sgRNA¸¦ ¹ßÇöÇÏ´Â CRISRP/Cas9 plasmid¸¦ Gesicle Á¦ÀÛ¿ë ¼¼Æ÷ÀÎ Gesicle Producer 293T Cell Line (Code 632617)¿¡ transfectionÇÏ¿© Cas9/sgRNA GesicleÀ» Á¦ÀÛÇÑ´Ù.

Cas9/sgRNA Gescle »ý¼º¿¡ ÇÊ¿äÇÑ ½Ã¾àÀº 2°³ÀÇ Æ©ºê¿¡ µ¿°á°ÇÁ¶ »óÅ·ΠÁ¦°øµÇ¸ç, sgRNA¸¦ Ŭ·Î´×ÇÑ pGuide-it sgRNA1 Vector¸¦ Æ©ºê¿¡ ÷°¡ ÈÄ, È¥ÇÕ¹°À» Gesicle Producer 293T Cell Line¿¡ transfectionÇÔÀ¸·Î½á °£´ÜÇÏ°Ô Cas9/sgRNA GesicleÀ» Á¦ÀÛÇÒ ¼ö ÀÖ´Ù.
CRISPR/Cas9 GesicleÀÇ ¿ø¸®
Guide-it CRISPR/Cas9 Gesicle Production SystemÀº °í³óµµÀÇ Cas9/sgRNA¸¦ Æ÷ÇÔÇÑ GesicleÀ» Á¦ÀÛÇÑ´Ù. Gesicle¿¡´Â Cas9 ´Ü¹éÁú, target specific sgRNA, CherryPicker Red Fluorosence protein, GlycoproteinÀ¸·Î ±¸¼ºµÇ¾îÀÖ´Ù.

¨ç sgRNA ¼­¿­À» »ðÀÔÇÑ pGuide-it-sgRNA1 vector¸¦ Gesicle Packaging Mix1°ú Mix2¸¦ ÀÌ¿ëÇÏ¿© Gesicle Producer 293T Cell Line¿¡ transfectionÇϸé Gesicle Producer 293T Cell membrane¿¡ GlycoproteinÀÌ »ý¼ºµÈ´Ù.
¨è ÀÌ ¶§, iDimerize ±â¼ú¿¡ ÀÇÇÏ¿© Cas9´Ü¹éÁú/sgRNA complex¿Í CherryPicker Çü±¤´Ü¹éÁúÀÌ Gesicle³»¿¡¼­ »óÈ£ÀÛ¿ëÇÏ°Ô µÇ¸ç, Gesicle ³»·Î ºÀÀÔ, ¼¼Æ÷ ¿Ü·Î ¹æÃâµÈ´Ù.
¨é ¹è¾ç¾×À¸·ÎºÎÅÍ È¸¼öÇÑ GeiscleÀ» ¸ñÀû¼¼Æ÷¿¡ ÷°¡Çϸé GesicleÀÌ ¼¼Æ÷¸·°ú À¶ÇյǾî Cas9/sgRNA Complex°¡ ¸ñÀû¼¼·Î ³»ºÎ·Î ¹æÃâµÈ´Ù.
¨ê NLS Tagging¿¡ ÀÇÇÏ¿© Cas9/sgRNA Complex´Â ¸ñÀû¼¼Æ÷ÀÇ ÇÙ ³»ºÎ·Î À̵¿Çϸç, ÇÙ ³»¿¡¼­ Genome DNAÀÇ target geneÀ» Àý´ÜÇÑ´Ù. ÀÌ ¶§, Gesicle¿¡ Æ÷ÇԵǾîÀÖ´Â CherryPicker Çü±¤´Ü¹éÁú·Î Cas9/sgRNA complexÀÇ ¼¼Æ÷ ³» µµÀÔÀ» ¸ð´ÏÅ͸µÇÒ ¼ö ÀÖ´Ù.


±×¸² 1. Cas9/sgRNA GesicleÀÇ Á¦ÀÛ°ú ¼¼Æ÷ ³» µµÀÔ ¿ø¸®


±×¸² 2. Cas9/sgRNA Gesicle¿¡ ÀÇÇÑ ´Ù¾çÇÑ ¼¼Æ÷ÀÇ Knock-out È¿À²

Cas9/sgRNA GesicleÀ» ÀÌ¿ëÇÏ¿© °¢ ¼¼Æ÷ÁÖ¿¡¼­ Zs-Green1À» Knock-outÇÏ¿´´Ù. ZsGreen1 Çü±¤´Ü¹éÁúÀ» ¸ñÀûÀ¸·Î Á¦ÀÛµÈ Cas9/sgRNA Gesicle ó¸® (°¢°¢ 10ul, 20ul, 30ul ÷°¡) ¶Ç´Â CRISPR/Cas9 plasmid transfectionÇÏ¿© Knock-out È¿À²À» ºñ±³ÇÏ¿´´Ù. Cas9/sgRNA GesicleÀ» ÀÌ¿ëÇÒ °æ¿ì transfection È¿À²ÀÌ ³·Àº ¼¼Æ÷¿¡ ´ëÇÏ¿© ÀÏÁ¤ÇÑ Á¦°Å È¿À²À» ¾òÀ» ¼ö ÀÖ¾ú´Ù.


±×¸² 3. Cas9-sgRNA gesicleÀ» »ç¿ëÇÑ endogenous protein(CD81) Knock-out


±×¸² 4. hiPS ¼¼Æ÷¿¡¼­ÀÇ CD81 Knock-out
Human CD81À» Ÿ°ÙÀ¸·Î µðÀÚÀÎµÈ Cas9-sgRNA complex¸¦ Æ÷ÇÔÇÑ Gesicles¸¦ ȸ¼ö ÈÄ, Cellartis human iPS cell line 18 (ChiPSC18) Kit (Code Y00305)¿¡ ó¸®ÇÏ°í Cellartis DEF-CS Culture System (Code Y30010) Á¸Àç ÇÏ¿¡ °¢°¢ 6½Ã°£, 24½Ã°£µ¿¾È ¹è¾çÇÏ¿´´Ù. ±× ÈÄ, Gesicle-free DEF-CS culture media·Î ¹èÁö ±³È¯ÇÏ¿© 7ÀÏ°£ Ãß°¡ ¹è¾çÇÏ¿´´Ù.
Gesicle-treated cell°ú untreated (control) cellÀÇ CD81 ¹ßÇöÀ²À» CD81À» Ÿ°ÙÇÏ´Â FITC-labeled antibodies¸¦ ÀÌ¿ëÇÏ¿© flowcytometry·Î ÃøÁ¤ÇÏ¿´´Ù.
Panel A. DEF-hiPSC ChiPSC18 cellÀÇ CD81 negative(¿ÞÂÊ) control°ú positive(¿À¸¥ÂÊ) control.
Panel B. Gesicle ó¸® 6½Ã°£(¿ÞÂÊ), 24½Ã°£ (¿À¸¥ÂÊ) ÈÄ, DEF-hiPSC ChiPSC18 cellÀÇ CD81 ¹ßÇöÀ²

* iPS ¼¼Æ÷ÀÇ CRISPR/Cas9 gene editing¿¡ ´ëÇÑ ÀÚ¼¼ÇÑ ³»¿ëÀº ¾Æ·¡¸¦ Ŭ¸¯ÇÏ¿© È®ÀÎÇϼ¼¿ä.
Footprint-free gene editing using CRISPR/Cas9 and single-cell cloning of edited human iPS cells
Àû¿ë
  • CRISPR/Cas9-mediated gene editing
  • Stem cell research
±¸¼ºÇ°
Guide-it CRISPR/Cas9 Gesicle Production System (Code 632613)
1.

Guide-it CRISPR/Cas9 Gesicle Packaging Set (Code 632616)

Guide-it CRISPR/Cas9 Gesicle Packaging Mix 1(green cap)
Guide-it CRISPR/Cas9 Gesicle Packaging Mix 2(yellow cap)
A/C Heterodimerizer(500 nM in Ethanol)
Protamine Sulfate(4 mg/ml)

10 vials
10 vials
50¥ì l
200¥ì l

2.

Guide-it sgRNA Vector System (Code 632612)

a. pGuide-it-sgRNA1 Vector(Linear)(7.5 ng/¥ì l)

12¥ì l

¡¡

b. Guide-it Ligation Components v2

DNA Ligation Mighty Mix
Guide-it Oligo Annealing Buffer
Guide-it Control Annealed Oligos v2(100 fmol/¥ì l)
Guide-it Sequencing Primer 1(100 pmol/¥ì l)
PCR-Grade Water

50¥ì l
1.5 ml
10¥ì l
10¥ì l
1 ml

¡¡

c. Stellar Competent Cells (Code 636763)

Stellar Competent Cells(100¥ì l/tube)
SOC Medium(1 ml/tube)
pUC19 Vector(0.1 ng/¥ì l)

10 tubes
10 tubes
10¥ì l

Gesicle Producer 293T Cell Line (Code 632617)

Gesicle Producer 293T Cell Line

1 ml

º¸Á¸
Stellar Competent Cells:-80¡É
±âŸ: -20¡É

Gesicle Producer 293T Cell Line: ¾×üÁú¼Ò(-196¡ÆC) ¶Ç´Â -150¡É freezer¿¡ º¸°ü
Cell Line Ãë±Þ ÁÖÀÇ»çÇ× (Ŭ¸¯)




Keyword : RNP,RibonucleoproteinµµÀÔÈ¿À²,microvesicle,iDimerize,CRISPR,CRISPR/Cas9,Cas9,sgRNA,Å©¸®½ºÆÛ,gene editing,À¯ÀüÀÚÆíÁý

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