Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system.
Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan S, Shivalila CS, Dadon DB, Jaenisch R
Cell Res. 2013 Aug 27. doi: 10.1038/cr.2013.122.
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PubMed
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Article
CRISPR-on is an RNA-guided transcriptional activator system with two components, the dCas9 activator protein and the single guide RNA (sgRNA). While the dCas9 protein provides the transactivation activity, the sgRNA component provides the specificity and is designed to target promoters or enhancers of genes of interest.
Different flavors of activators were constructed, with VP48 containing 3x minimal VP16 domains, VP64 with 4, VP96 with 6, and VP160 with 10.
dCas9 activators and sgRNAs can be separately transfected (pmax dCas9 activators (#48223–48227) + sgRNA expression vector or the linear DNA containing the sgRNA expressing fragment produced by PCR), or expressed from a single dual-expression vector (#48236–48240).
dCas9 activator ORFs (with Stop codon) are also available as Gateway donor vectors (#48218–48221) and can be transferred to Gateway destination vectors (e.g., pmax-DEST #48222) and Lifetechnologies Gateway Destination Vectors (Link opens in a new window)) to adapt to different systems by LR clonase reaction.
dCas9 Activators sgRNA Dual Expression
| ID | Plasmid |
|---|---|
| 48236 | pAC2-dual-dCas9VP48-sgExpression: Dual expression construct expressing both dCas9VP48 and sgRNA from separate promoters. |
| 48238 | pAC152-dual-dCas9VP64-sgExpression: Dual expression construct expressing both dCas9VP64 and sgRNA from separate promoters. |
| 48239 | pAC153-dual-dCas9VP96-sgExpression: Dual expression construct expressing both dCas9VP64 and sgRNA from separate promoters. |
| 48240 | pAC154-dual-dCas9VP160-sgExpression: Dual expression construct expressing both dCas9VP64 and sgRNA from separate promoters. |
pmax dCas9 Activator Expression Plasmids
| ID | Plasmid |
|---|---|
| 48223 | pAC91-pmax-dCas9VP64: dCas9VP64 on pmax expression vector. |
| 48224 | pAC92-pmax-dCas9VP96: dCas9VP96 on pmax expression vector. |
| 48225 | pAC93-pmax-dCas9VP160: dCas9VP160 on pmax expression vector. |
| 48226 | pAC94-pmax-dCas9VP160-2A-puro: dCas9VP160-2A-puro (puro-selectable) on pmax expression vecor. |
dCas9 Activators Gateway Donors
| ID | Plasmid |
|---|---|
| 48214 | pAC1-pCR8-dCas9VP48: dCas9VP48 on Gateway donor vector pCR8/GW/TOPO. |
| 48218 | pAC84-pCR8-dCas9: dCas9 on Gateway donor vector pCR8/GW/TOPO. |
| 48219 | pAC147-pCR8-dCas9VP64: dCas9VP64 on Gateway donor vector pCR8/GW/TOPO. |
| 48220 | pAC148-pCR8-dCas9VP96: dCas9VP96 on Gateway donor vector pCR8/GW/TOPO. |
| 48221 | pAC149-pCR8-dCas9VP160: dCas9VP160 on Gateway donor vector pCR8/GW/TOPO. |
Gateway Destination Vector
| ID | Plasmid |
|---|---|
| 48222 | pAC90-pmax-DEST: Gateway Destination vector derived from Clontech's pmax (CAGGS) expression vector. |
Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 48214 | pAC1-pCR8-dCas9VP48 | dCas9VP48 on Gateway donor vector pCR8/GW/TOPO. Note: This is not for expression. It has to be transferred to a gateway destination vector for expression |
| 48218 | pAC84-pCR8-dCas9 | dCas9 on Gateway donor vector pCR8/GW/TOPO. Note: This is not for expression. It has to be transferred to a gateway destination vector for expression |
| 48219 | pAC147-pCR8-dCas9VP64 | dCas9VP64 on Gateway donor vector pCR8/GW/TOPO. Note: This is not for expression. It has to be transferred to a gateway destination vector for expression |
| 48220 | pAC148-pCR8-dCas9VP96 | dCas9VP96 on Gateway donor vector pCR8/GW/TOPO. Note: This is not for expression. It has to be transferred to a gateway destination vector for expression |
| 48221 | pAC149-pCR8-dCas9VP160 | dCas9VP160 on Gateway donor vector pCR8/GW/TOPO. Note: This is not for expression. It has to be transferred to a gateway destination vector for expression |
| 48222 | pAC90-pmax-DEST | Gateway Destination vector derived from Clontech's pmax (CAGGS) expression vector |
| 48223 | pAC91-pmax-dCas9VP64 | dCas9VP64 on pmax expression vector |
| 48224 | pAC92-pmax-dCas9VP96 | dCas9VP96 on pmax expression vector |
| 48225 | pAC93-pmax-dCas9VP160 | dCas9VP160 on pmax expression vector |
| 48226 | pAC94-pmax-dCas9VP160-2A-puro | dCas9VP160-2A-puro (puro-selectable) on pmax expression vecor. Note: This is being tested. |
| 48227 | pAC95-pmax-dCas9VP160-2A-neo | dCas9VP160-2A-neo (neo/G418-selectable) on pmax expression vector. Note: This is being tested. |
| 48228 | pAC103-PBNeo-U6-sgRNA-Expression | sgRNA expression on PiggyBac vector with Neo-selectable marker |
| 48234 | pAC150-PBLHL-4xHS-EF1a-DEST | PiggyBac expression gateway destination vector with Insulator-flanked EF1a promoter gateway cassette and floxed Hygro selection marker |
| 48236 | pAC2-dual-dCas9VP48-sgExpression | Dual expression construct expressing both dCas9VP48 and sgRNA from separate promoters |
| 48237 | pAC5-dual-dCas9VP48-sgTetO | Dual expression construct expressing both dCas9VP48 and sgTetO from separate promoters |
| 48238 | pAC152-dual-dCas9VP64-sgExpression | Dual expression construct expressing both dCas9VP64 and sgRNA from separate promoters |
| 48239 | pAC153-dual-dCas9VP96-sgExpression | Dual expression construct expressing both dCas9VP96 and sgRNA from separate promoters |
| 48240 | pAC154-dual-dCas9VP160-sgExpression | Dual expression construct expressing both dCas9VP160 and sgRNA from separate promoters |
| 49045 | pAC155-pCR8-sgExpression | U6-driven sgRNA expressing cassette on a gateway donor vector |