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Lentivirus¸¦ ÀÌ¿ëÇÑ sgRNA, Cas9 gene delivery

Lenti-X¢â CRISPR/Cas9 System

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Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Clontech
632629
Lenti-X¢â CRISPR/Cas9 System
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç°  ¶óÀ̼±½º 
Each
(Package)
2,211,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32765
Clontech
632630
pLVX-hyg-sgRNA1 Vector System
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç°  ¶óÀ̼±½º 
10 Rxns
(Package)
592,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32765
Clontech
632633
Lenti-X¢â Tet-On¢ç 3G CRISPR/Cas9 System
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç°  ¶óÀ̼±½º 
1 System
(Package)
3,562,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32785

  • Cas9 gene°ú sgRNA¸¦ Lentivirus¸¦ ÀÌ¿ëÇÏ¿© ¼¼Æ÷ ³» µµÀÔ
  • TransfectionÀÌ ¾î·Á¿î mammalian ¼¼Æ÷ (proliferating and non-proliferating cells)¿¡¼­ÀÇ CRISPR/Cas9 Genome editing
  • Lenti-X¢â Packaging Single shotsÀ» ÀÌ¿ëÇÏ¿© °£´ÜÇÏ°Ô °í¿ª°¡ÀÇ Lentivirus Á¦ÀÛ
  • Lenti-X¢â Tet-On 3G CRISPR/Cas9 systemÀº Cas9 ¹ßÇöÀ» È¿À²ÀûÀ¸·Î Á¶ÀýÀÌ °¡´ÉÇϸç, ¼¼Æ÷ µ¶¼º ¹®Á¦³ª Cas9ÀÇ Áö¼ÓÀûÀÎ ¹ßÇöÀ¸·Î ÀÎÇÑ off-target effect °¨¼Ò¿¡ È¿°úÀû
Á¦Ç°¼³¸í
Lenti-X¢â CRISPR/Cas9 SystemÀº lentiviral-mediated CRISPR/Cas9 genome editingÀ» À§ÇÑ ¸ðµç ±¸¼ºÇ°ÀÌ Æ÷ÇÔµÈ complete systemÀÌ´Ù.
º» Á¦Ç°Àº °í¿ª°¡ÀÇ ·»Æ¼¹ÙÀÌ·¯½º¸¦ »ý»êÇÏ´Â Lenti-X¢â Packaging Single Shots°ú Cas9 ´Ü¹éÁú, sgRNA¸¦ ¹ßÇöÇÏ´Â µÎ °³ÀÇ Çö󽺹̵å·Î ±¸¼ºµÇ¾îÀÖ´Ù. ¸ñÀû À¯ÀüÀÚ¸¦ Ÿ°ÙÇÏ´Â sgRNA¸¦ ¹ßÇöÇÏ´Â pLVX-hgy-sgRNA1 plasmid´Â ÀÌ¹Ì ¼±ÇüÈ­ µÇ¾îÀÖ¾î ¾ÆÁÖ ½±°Ô sgRNA ¼­¿­À» »ðÀÔ, ¶óÀÌ°ÔÀ̼ÇÀ» ÁøÇàÇÒ ¼ö ÀÖ´Ù. Lentivirus¸¦ ÀÌ¿ëÇÏ¿© sgRNA¿Í Cas9 geneÀ» »ðÀÔÇϸé ÀϹÝÀûÀ¸·Î TransfectionÀÌ ¾î·Æ´Ù°í ¾Ë·ÁÁø ¼¼Æ÷ÁÖ¿¡¼­ÀÇ CRISPR/Cas9 genome editingÀÌ °¡´ÉÇÏ´Ù (±×¸² 1).

Lenti-X¢â Tet-On 3G CRISPR/Cas9 SystemÀº doxycyclineÀ» ÀÌ¿ëÇÏ¿© Cas9 ¹ßÇöÀ» À¯µµÇÒ ¼ö ÀÖ´Â Tet-on 3G inducible expression Á¦Ç°À¸·Î½á, doxycycline ó¸® ½Ã Cas9 ´Ü¹éÁúÀ» ¹ßÇöÇÑ´Ù. Cas9 ¹ßÇöÀ» ¾ö°ÝÇÏ°Ô Á¶ÀýÇÒ ¼ö Àֱ⠶§¹®¿¡ ¿µ±¸ÀûÀÎ Cas9 ´Ü¹éÁú ¹ßÇöÀ¸·Î ÀÎÇÑ off-target effect³ª ¼¼Æ÷ µ¶¼ºÀ» ÁÙÀÏ ¼ö ÀÖ´Ù (±×¸² 2, ±×¸² 3).


±×¸² 1. Either Jurkat or HT1080 cells were first transduced with LVX-hyg-CD81-sgRNA and then selected for stable integration using hygromycin. Stable clones were then transduced with LVX-puro-Cas9. Stable clones were selected for using puromycin and then screened for CD81 knockout efficiency using FACS. Positive and negative controls of parental cells without Cas9 transduction were done either with or without anti-CD81 antibody.


±×¸² 2. Inducible knockout of CD81 in HEK293 cells. Target cells were first transduced with Tet-On 3G lentivirus and selected with G418. Cas9 induction was then assayed by Western blot using polyclonal anti-Cas9 antibody (Code 632607), diluted 1:1,000, and ECL reagent. Panel A. Western blot results for six independent Tet-On 3G-Cas9-positive cell populations cultured in the absence (-) or presence (+) of 0.5 ¥ìg/ml doxycycline, assayed with anti-Cas9 antibody. Panel B. FACS results for three independent Tet-On 3G-Cas9-positive 293T cell populations transduced with CD81-sgRNA lentivirus and cultured in the absence (-Dox) or presence (+Dox) of 0.5 ¥ìg/ml doxycycline.


±×¸² 3. Inducible editing of AAVS1in HEK293 cells. Induction of Cas9 expression in six stable Tet-On 3G-Cas9-positive HEK293 cell lines was assayed by qRT-PCR. The Guide-it Mutation Detection Kit (Code 631443) was then used to screen for editing of AAVS1. Panel A. qRT-PCR results for Cas9 expression in uninduced (-Dox) and induced (+Dox) cells for each clone. Panel B. Results of Guide-it resolvase assay for detection of AAVS1 gene editing in uninduced (?) and induced (+) clones.
Applications
Lentivirus-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)
Lenti-X CRISPR/Cas9 System (Code 632629)

pLVX-hyg-sgRNA1 Vector System (Code 632630)

Lenti-X¢â Tet-On 3G CRISPR/Cas9 System (Code 632633)




Keyword : Lenti,Lentivirus,Lentiviral,Tet,Tetracycline,inducible,inducible system,µµÀÔÈ¿À²,CRISPR,CRISPR/Cas9,Cas9,sgRNA,Å©¸®½ºÆÛ,gene editing,À¯ÀüÀÚÆíÁý

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