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CRISPR header icon 适用于基因组工程的: CRISPR/Cas


CRISPR - Cas9基因组编辑技术已成为一个重要的基因工程工具 . Cas9酶已经被开发来执行各种任务(截, 修, 干扰, 放大, 提纯) . 质粒的下面的一组用于创建在不同物种定制CRISPR目标

CRISPR-Cas9-for-Genome-Engineering-Grey-Background-Scissors.png
  1. 选择应用程序的CRISPR/ CAS系统:

    • - 使用Cas9引入双链断裂(DNA双链断裂)和非同源末端连接(NHEJ)进行维修 .
    • - 编辑的DNA序列通过使用“切口酶'Cas9和同源捐助模板通过诱导同源定向修复(HDR)的途径。 .
    • 干扰 - 击倒的基因的表达通过使用催化活性的Cas9干扰转录 .
    • 激活 - 通过催化无效cas9的方式增加基因表达 .
    • 提纯 - 分离的基因组中的特定区域用使用催化无效,标有Cas9 .

  2. 接下来,选择适当的引导RNA(gRNA):

    • 在列以的右边cas9质粒,进行验证gRNAs列 . 要针对你的基因组中的感兴趣区域,复制您的自定义寡核苷酸进入这个gRNA表达盒。 .

截:Cas9核酸酶

全功能Cas9酶的设计理念引入双链断裂(DNA双链断裂)在一个特定的位置,基于gRNA靶序列。 NHEJ修复DNA双链断裂的细胞. NHEJ导致移码或提前终止密码子,由于插入或缺失(个InDel) . 移码和过早终止密码子干扰DNA序列。

   Cas9 Plasmid Promoter Validated in Delivery Selection Co-published
gRNA Plasmid,
if separate
Depositing lab
Cas9 species = S. pyogenes (PAM = NGG)
M-SPcas CMV 293T Transfection Neo/G418 gRNA_Cloning Vector Church
hCas9 CMV 293T, K562,
iPS (PGP1)
Transfection Neo/G418 gRNA_Cloning Vector Church
pMJ920 CMV 293T Transfection Neo/G418
EGFP
pSilencer 2.1-U6 puro
(Life Tech)
Doudna
pCAG-T3-hCAS-pA CAG Embryo Transfection In vitro transcription
→ microinjection
Fujii
pCAG-hCas9 CAG human iPSCs Transfection Neo/G418 need separate gRNA plasmid Hatada
pST1374 CMV Embryo Transfection Blasticidin In vitro transcription
→ transfection of ssRNA
Huang
JDS246 CMV Transfection MLM3636 Joung
p3s-Cas9HC CMV 293T Transfection In vitro transcription
→ transfection of ssRNA
Kim
pCas9_GFP CAG hPSCs Transfection EGFP gRNA_Cloning Vector Musunuru
pCW-Cas9 Tet ON 293T Lentiviral Puromycin pLX-sgRNA Sabatini and
Lander
lentiCRISPR v2 EFS A375, HUES62 Lentiviral Puromycin Contained on
same plasmid
Zhang
lentiCas9-Blast EFS A375, HUES62 Lentiviral Blasticidin lentiGuide Puro Zhang
PX459 (3rd Gen) CBh 293T, hESCs Transfection Puromycin Contained on
same plasmid
Zhang
PX458 (3rd Gen) CBh 293T, hESCs Transfection EGFP Contained on
same plasmid
Zhang
PX330 (2nd Gen) CBh 293T, hESCs,
Mouse neuron (N2A)
Transfection Contained on
same plasmid
Zhang
PX260 (1st Gen) CBh 293T, hESCs,
Mouse neuron (N2A)
Transfection Puromycin Contained on
same plasmid
Zhang
PX165 (1st Gen) CBh 293T, hESCs Transfection Zhang
Cas9 species = N. meningitidis (PAM = NNNNGATT)
M-NMcas CMV 293T Transfection Neo/G418 see paper Church
pSimpleII-NmCas9-FLAG EF1a human iPSCs,
hESCs, 293FT
Transfection pSmart-Nm-sgRNA-BbsI Thomson and
Sontheimer
pSimpleII-NLS-NmCas9-HA-NLS(s) EF1a human iPSCs,
hESCs, 293FT
Transfection pSmart-Nm-sgRNA-BbsI Thomson and
Sontheimer
pSimpleII-U6-tracr-
U6-BsmBI-NLS-
NmCas9-HA-NLS(s)
EF1a human iPSCs,
hESCs, 293FT
Transfection Contained on same plasmid Thomson and
Sontheimer
Cas9 species = S. thermophilus (PAM = NNAGAA)
M-ST1cas CMV 293T Transfection Neo/G418 see paper Church
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修:Cas9切口酶

突变Cas9酶(通常D10A或H840A)设计为切断对DNA的一条链在特定的位置的基础上gRNA靶序列。编辑的DNA序列通过使用“切口酶'Cas9和同源捐助模板通过诱导同源定向修复(HDR)的途径。在细胞中的DNA缺口被同源定向修复(HDR)的修复。 HDR可以用来忠实地引入特定的核苷酸变化,是不易出错比NHEJ。

验证物种 验证子物种或细胞类型 促进者 实验室CRISPR页(更多信息 PAM序列 Cas9质粒 gRNA质粒
Human 293T, K562, iPS (PGP1) CMV Church NGG hCas9 D10A gRNA_Cloning Vector
293T, hESC CBh Zhang NGG pX335, pX334, pSpCas9n(BB)-2A-GFP (PX461), pSpCas9n(BB)-2A-Puro (PX462), pSpCas9n(BB) (PX460) Dual expression Cas9 & gRNA on same plasmid
Pluripotent stem cells (hPSCs) CAG Musunuru NGG pCas9D10A_GFP gRNA_Cloning Vector
Mouse Mouse neuron (N2A) CBh Zhang NGG pX335, pX334 Dual expression Cas9 & gRNA on same plasmid
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干扰: CRISPR干扰(CRISPRi)

催化无效Cas9(dCas9)被称为“dCas9转录阻遏蛋白融合”,针对基于gRNA靶序列的特定位置。 非活动Cas9扰乱转录和基因的表达。典型目标地点,包括启动子区或编码区。

验证物种 验证子物种或细胞类型 促进者 实验室CRISPR页(更多信息 PAM序列 Cas9质粒 gRNA质粒
Bacteria DH5alpha, E. coli, S. pneumoniae DB17 tet promoter Marraffini NGG pdCas9 Dual expression Cas9 & gRNA on same plasmid
tet promoter Qi NGG pdCas9-bacteria pgRNA-bacteria
Human 293T MSCV LTR promoter Qi NGG pdCas9-humanized pgRNA-humanized
293T SFFV Qi-Weissman NGG pHR-SFFV-dCas9-BFP, pHR-SFFV-dCas9-BFP-KRAB pgRNA-humanized
293T CMV Church NGG Cas9m2, Cas9m3, Cas9m4 gRNA_Cloning Vector
293T CMV Gersbach NGG pcDNA-dCas9 pSPgRNA
Yeast S. cerevisiae TDH3 Qi-Weissman NGG pTDH3-dCas9, pTDH3-dCas9-Mxi1 -
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激活:dCas9激活因子融合

催化无效Cas9(dCas9)被称为“dCas9转录阻遏蛋白融合”,针对基于gRNA靶序列的特定位置。 非活动Cas9扰乱转录和基因的表达。典型目标地点,包括启动子区或编码区。
验证物种 验证子物种或细胞类型 促进者 实验室CRISPR页(更多信息 PAM序列 Cas9质粒 gRNA质粒
Human 293T CMV Church NGG Cas9m2-VP64, Cas9m3-VP64, Cas9m4-VP64 gRNA_Cloning Vector
293T CMV Joung NGG pSL690, pMLM3705, pMLM3636
293T CMV Gersbach NGG pcDNA-dCas9-VP64 pSPgRNA
293T, HeLa CAGGS Jaenisch NGG pAC94-pmax-dCas9VP160-2A-puro, pAC95-pmax-dCas9VP160-2A-neo pAC155-pCR8-sgExpression
293T, HeLa CBh Jaenisch NGG pAC152-64Dual, pAC153-96Dual, pAC154-160Dual, pAC2-dual-dCas9VP48-sgExpression, pAC5-dual-dCas9VP48-sgTetO Dual expression Cas9 & gRNA on same plasmid
CAGGS Jaenisch NGG CAGGS-64, CAGGS-96, CAGGS-160 Transfection of PCR product coding gRNA
- - Jaenisch NGG dCas9VP64, dCas9VP96, dCas9VP160 -
293T MSCV LTR Qi-Weismann NGG pMSCV-LTR-dCas9-p65AD-BFP, pMSCV-LTR-dCas9-VP64-BFP pgRNA-humanized
293T CMV Lu NGG pCMV_dCas9_VP64 pU6_gRNA_handle_U6t
Mouse NIH3T3, mESC, mouse embryos CBh Jaenisch NGG pAC152-64Dual, pAC153-96Dual, pAC154-160Dual Dual expression Cas9 & gRNA on same plasmid
MEF CMV Gersbach NGG pcDNA-dCas9-VP64 pSPgRNA
NIH3T3, mESC, mouse embryos CAGGS Jaenisch NGG CAGGS-64, CAGGS-96, CAGGS-160 Transfection of PCR product coding gRNA
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