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CRISPR Knock-outÀ» ÀÌ¿ëÇÑ 19,000°³ ÀÌ»óÀÇ À¯ÀüÀÚ Ç¥ÇöÇü ½ºÅ©¸®´×

Genome-Wide sgRNA Library System

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Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
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Clontech
632646
Guide-it¢â CRISPR Genome-Wide sgRNA Library System
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç°  ¶óÀ̼±½º 
5 Screens
(Package)
3,074,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x98810, x98812
Clontech
632647
Guide-it¢â CRISPR Genome-Wide sgRNA Library NGS Analysis Kit
°ü·ÃÇмú ±¸¸ÅÇϱâ
10 ȸ
(Package)
1,229,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x98810
Clontech
632651
Guide-it¢â CRISPR Genome-Wide Library PCR Kit
°ü·ÃÇмú ±¸¸ÅÇϱâ
20 ȸ
534,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x98810

  • Brunello library ±â¹ÝÀÇ 76,000°³ÀÇ sgRNA Æ÷ÇÔ (Download sgRNA representation data.)
    (Brunello library : on-target ºñÀ²Àº ±Ø´ëÈ­ÇÏ°í off-target effect´Â ÃÖ¼ÒÈ­½ÃŲ sgRNA library)
  • Human GenomeÀÇ °¢ À¯ÀüÀÚ¸¶´Ù 4°³ÀÇ sgRNA°¡ Ÿ°Ù
  • º¯Çü sgRNA scaffold µðÀÚÀÎÀ» Àû¿ëÇÏ¿© Cas9-sgRNA »óÈ£ÀÛ¿ëÀ» º¸´Ù °³¼±½ÃÅ°°í À¯ÀüÀÚÆíÁý ÆíÁý È¿À² Çâ»ó
  • Lentiviral sgRNA library´Â Lenti-X Packaging Single Shots ÇüÅ·ΠÁ¦ÀÛµÇ¾î °í¿ª°¡ÀÇ Lentivirus sgRNA library¸¦ ¼Õ½±°Ô Á¦ÀÛ °¡´É
  • ¿¬±¸ÀÚÀÇ ¾ÈÀü(safety)À» À§ÇÑ Self-inactivating lentiviral vector Àû¿ë
Á¦Ç°¼³¸í
Guide-it¢â CRISPR Genome-Wide sgRNA Library SystemÀº Àΰ£¼¼Æ÷¿¡¼­ CRISPR/Cas9 Genome EditingÀ» ÀÌ¿ëÇÑ Àüü À¯ÀüÀÚ Ç¥ÇöÇü ½ºÅ©¸®´× (Genome-wide phenotypic knock-out screening)À» ¿ëÀÌÇÏ°Ô ÇÒ ¼ö ÀÖµµ·Ï Á¦ÀÛµÈ Á¦Ç°ÀÌ´Ù. º» Á¦Ç°Àº Cas9°ú sgRNA library¸¦ ¾ÈÁ¤ÀûÀ¸·Î ¹ßÇöÇÏ´Â ¼¼Æ÷ÁÖ¸¦ ½±°Ô Á¦ÀÛÇÒ ¼ö ÀÖµµ·Ï Lenti-X Single Shots ÇüÅÂÀÇ genome-wide sgRNA library¿Í Cas9 ´Ü¹éÁú ¹ßÇö ½Ã½ºÅÛÀ» Æ÷ÇÔÇÏ°í ÀÖ´Ù. Genome-wide sgRNA library´Â Brunello library (Doench et al. 2016; Doench, 2018)¸¦ ±â¹ÝÀ¸·Î ÇÏ°í ÀÖÀ¸¸ç, À¯ÀüÀÚ Çϳª ´ç, sgRNA 4°³¾¿, ÃÑ >76,000°³ÀÇ sgRNA¸¦ Á¦°øÇÑ´Ù. ±×·¯¹Ç·Î ÃÑ 19,000°³ÀÇ À¯ÀüÀÚ¸¦ Ÿ°ÙÆÃÇÏ¿© Ç¥ÇöÇü ½ºÅ©¸®´×À» ÁøÇàÇÒ ¼ö ÀÖ´Ù. ½ºÅ©¸®´×À» ÁøÇà ÈÄ, Guide-it¢â CRISPR Genome-Wide sgRNA Library NGS Analysis Kit (Code 632647)À» ÀÌ¿ëÇϸé next-generation sequencing ºÐ¼®À» ÁøÇàÇÒ ¼ö ÀÖ´Ù. Guide-it¢â CRISPR Genome-Wide sgRNA Library NGS Analysis Kit (Code 632647) Á¦Ç° ³»¿¡´Â sgRNA library°¡ µµÀÔµÈ ¼¼Æ÷·ÎºÎÅÍ NGS library¸¦ Á¦ÀÛÇϱâ À§ÇÑ ¸ðµç ÇÁ¶óÀÌ¸Ó¿Í ½Ã¾àÀÌ Æ÷ÇԵǾîÀÖ´Ù. º» Á¦Ç°ÀÇ sgRNA library´Â sgRNA representationÀ» º¸ÀåÇÒ ¼ö ÀÖµµ·Ï Á¦À۵ǾúÀ¸¸ç, ´ÙÄ«¶ó¹ÙÀÌ¿À´Â library ÇÕ¼º, ÁõÆø, ¹ÙÀÌ·¯½º Á¦ÀÛ ¹× Ÿ°Ù ¼¼Æ÷ µµÀÔÀÇ °¢ ´Ü°è¿¡¼­ ¸ðµÎ sgRNA representationÀ» ÃøÁ¤ ¿Ï·áÇÏ¿´´Ù.
º» Á¦Ç°Àº µ¿°á°ÇÁ¶µÈ Lentivirus ÇüÅ (dry-down lentiviral format)ÀÇ validated sgRNA library¿Í Lenti-X 293T cellÀ» Æ÷ÇÔÇÏ°í ÀÖÀ¸¸ç, µ¿°á°ÇÁ¶»óÅÂÀÇ library mix¿¡ ¹°¸¸ ³Ö°í 293T Cell¿¡ ÷°¡ ¹× ¹è¾çÇϸé Lentiviral particleÀ» ¸¸µé ¼ö ÀÖµµ·Ï Á¦À۵Ǿî ÀÖ´Ù.


±×¸² 1. Vector and sgRNA scaffold design Guide-it CRISPR sgRNA library. 
Panel A. pLVXS-EF1a-Cas9-PGK-Puro and pLVXS-sgRNA-mCherry-hyg vectors map.
Panel B. sgRNA scaffold design. sgRNA´Â Human U6 promoter·ÎºÎÅÍ ¹ßÇöµÇ¸ç ÃÖÀûÈ­µÈ scaffold ¼­¿­Àº Cas9 loadingÀ» ¿ëÀÌÇÏ°Ô ÇÏ¿© genome editing È¿À²À» ³ô¿©ÁØ´Ù.


±×¸² 2. mCherry Çü±¤´Ü¹éÁúÀ» ÀÌ¿ëÇÑ sgRNA library lentivirusÀÇ µµÀÔÈ¿À² È®ÀÎ ¹× °áÁ¤
sgRNA library¸¦ Æ÷ÇÔÇÑ Lentivirus particleÀ» »ý»ê ÈÄ, Cas9À» ¹ßÇöÇÏ´Â A375 cell¿¡ ´Ù¾çÇÑ MOI·Î µµÀÔÇÏ¿´´Ù. 48½Ã°£ ÈÄ, FACS¿Í Çü±¤Çö¹Ì°æÀ» ÀÌ¿ëÇÏ¿© sgRNA library lentivirusÀÇ µµÀÔÈ¿À²À» È®ÀÎÇÏ¿´´Ù


±×¸² 3. Representation of sgRNAs within the Guide-it CRISPR Genome-Wide sgRNA Library System.
Panel A. Guide representation in plasmid library.
Panel B. Comparison of guide representation in the plasmid library and transduced cells.


±×¸² 4. Identification and analysis of sgRNAs isolated from cells after a 6-thioguanine (6-TG) screen.
Panel A. sgRNA representation was compared between the original plasmid library population used to make the lentivirus preparation and the resulting transduced, selected and non-selected cell populations. All four sgRNAs for HPRT (red dots) and NUDT5 (black dots) were enriched in the 6-TG-selected population (blue dots). The representation of these sgRNAs in the plasmid library (gray) and transduced, non-selected population (orange) is also shown.
Panel B. After selection, the sgRNA representation was shifted in response (blue) to 6-TG. Individual sgRNAs that were enriched are highlighted within this correlation (red and orange dots). Inset shows fold-enrichment of the four sgRNAs for the HPRT gene as compared to the non-selected population.


±×¸² 5. Workflow schematic for an sgRNA library screen using 6-thioguanine (6-TG) selection.
Àû¿ë
  • Pooled genome-wide phenotypic screens in human cells
  • Loss-of-function genetic screens
±¸¼ºÇ° (ÀÚ¼¼ÇÑ ³»¿ëÀº CoA¸¦ ÂüÁ¶Çϼ¼¿ä)
Guide-it¢â CRISPR Genome-Wide sgRNA Library System (Code 632646)
  • Guide-it Genome-Wide sgRNA Library Transfection Mix
  • Guide-it Cas9 Lentiviral Transfection Mix
  • pLVXS-sgRNA-mCherry-hyg Vector
  • Lenti-X 293T Cell Line
  • Lenti-X GoStix Plus (Sample-Not for Sale)

Guide-it¢â CRISPR Genome-Wide sgRNA Library NGS Analysis Kit (Code 632647)
  • Guide-it CRISPR Genome-Wide Library PCR Kit
  • NucleoBond CB 500
  • NucleoSpin Gel and PCR Clean-Up

Guide-it¢â CRISPR Genome-Wide Library PCR Kit (Code 632651)
  • LVXS P5 primer pool with index 1 (10 ¥ìM)
  • LVXS P7 primer with index 2 (10 ¥ìM)
  • LVXS P7 primer with index 3 (10 ¥ìM)
  • LVXS P7 primer with index 4 (10 ¥ìM)
  • LVXS P7 primer with index 5 (10 ¥ìM)
  • TaKaRa Ex Taq¢ç (5 U/¥ìl)
  • 10X Ex Taq¢â Buffer (Mg2+ plus)
  • dNTP Mixture (2.5 mM each)
Notes
  • Guide-it¢â CRISPR Genome-Wide Library PCR Kit (Code 632651) Á¦Ç° ³»¿¡´Â NGS ºÐ¼®À» À§ÇÑ genomic DNA ÃßÃâ, sgRNA ¼­¿­ ÁõÆøÇÏ´Â µ¥ ÇÊ¿äÇÑ ÇÁ¶óÀÌ¸Ó¿Í ½Ã¾àÀ» Æ÷ÇÔÇÏ°í ÀÖ´Ù. º» Á¦Ç°À» ÀÌ¿ëÇÏ¿© 10¹øÀÇ sequencing library Á¦ÀÛÀÌ °¡´ÉÇÏ´Ù (20 PCRs)
  • Guide-it¢â CRISPR Genome-Wide sgRNA Library NGS Analysis Kit (Code 632647) Á¦Ç°Àº Guide-it¢â CRISPR Genome-Wide Library PCR Kit (Code 632651)¿Ü, genomic DNA ÃßÃâ ¹× PCR »ê¹° Á¤Á¦ Å°Æ®°¡ Æ÷ÇԵǾîÀÖ´Ù.




Keyword : Brunello,Lentivirus,sgRNA library,Knockout,knock out screening,gene screening,phenotype,array,NGS,CRISPR,CRISPR/Cas9,Cas9,sgRNA,Å©¸®½ºÆÛ,gene editing,À¯ÀüÀÚÆíÁý

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