dCas9 regulator to neutralize competition in CRISPRi circuits
crispr-on - AddgeneCRISPR-on is an RNA-guided Transcriptional activator system with two compnents,...
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dCas9 Activator AttL1 AttL2 dCas9 Gateway Donor Series AttR2 AttR1 Gateway Destination Vectors, e.g., pmax-DEST pmax dCas9 Activators dCas9 Activator CAGGS Dual dCas9 Activator sgRNA Expression dCas9 Activator sgRNA sgRNA PCR sgRNA PiggyBac sgRNA Expression Plasmids PCR ... PiggyBac dCas9 Activator expression Plasmids EF1a dCas9 Activator Not Available on Addgene due to licensing issues of the PiggyBac technology Activator Plasmid Addgene ID Selection VP64 pAC91 48223 - VP96 pAC92 48224 - VP160 pAC93 48225 - VP160 pAC94 48226 (2A)-Puro VP160 pAC95 48227 (2A)-Neo Activator Plasmid Addgene ID - pAC84 48218 VP48 pAC1 48214 VP64 pAC147 48219 VP96 pAC148 48220 VP160 pAC149 48221 Promoter Plasmid Addgene ID CAGGS pAC90 48222 3. dCas9 Activators Gateway Donors 4. Gateway Destination vector 2. pmax dCas9 Activator Expression plasmids Activator Plasmid Addgene ID VP48 pAC2 48236 VP64 pAC152 48238 VP96 pAC153 48239 VP160 pAC154 48240 1. dCas9 Activators sgRNA Dual Expression CRISPR-on is an RNA-guided Transcriptional activator system with two compnents, 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 trans- fected (pmax dCas9 activators [Tabe 2] + sgRNA expresssion vector or the linear DNA containing the sgRNA expressing fragment produced by PCR), or expressed from a single dual-expression vector [Table 1]. dCas9 activator ORFs (with Stop codon) are also ava- ialble as Gateway donor vectors [Table 3] and can be transferred to Gateway destination vectors (e.g., pmax-DEST [Table 4] and and http://www.lifetechnologies.com/us/en/home/life- science/cloning/gateway-cloning/gateway-destination- vectors.html) to adapt to different systems by LR clon- ase reaction. crispr-on.org “Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system” Cell Research doi: 10.1038/cr.2013.122
Transcript of crispr-on - AddgeneCRISPR-on is an RNA-guided Transcriptional activator system with two compnents,...
dCas9Activator
AttL1 AttL2
dCas9 Gateway Donor Series
AttR2AttR1
Gateway DestinationVectors, e.g.,pmax-DEST
pmaxdCas9 Activators
dCas9 Activator
CAGG
S
DualdCas9 Activator
sgRNA Expression
dCas9Activator
sgRNA
sgRNA
PCR
sgRNA
PiggyBacsgRNA Expression
Plasmids
PCR
...
PiggyBacdCas9 Activator
expressionPlasmids
EF1a
dCas9 Activator
Not Available on Addgene due to licensing issues of the PiggyBac technology
Activator Plasmid Addgene ID SelectionVP64 pAC91 48223 -VP96 pAC92 48224 -
VP160 pAC93 48225 -VP160 pAC94 48226 (2A)-PuroVP160 pAC95 48227 (2A)-Neo
Activator Plasmid Addgene ID- pAC84 48218
VP48 pAC1 48214VP64 pAC147 48219VP96 pAC148 48220
VP160 pAC149 48221
Promoter Plasmid Addgene IDCAGGS pAC90 48222
3. dCas9 Activators Gateway Donors
4. Gateway Destination vector
2. pmax dCas9 Activator Expression plasmids
Activator Plasmid Addgene IDVP48 pAC2 48236VP64 pAC152 48238VP96 pAC153 48239
VP160 pAC154 48240
1. dCas9 Activators sgRNA Dual Expression
CRISPR-on is an RNA-guided Transcriptional activator system with two compnents, the dCas9 activator protein and the single guide RNA (sgRNA). While the dCas9 protein provides the transactivation activity, the sgRNA component provides the speci�city and is designed to target promoters or enhancers of genes of interest.
Di�erent �avors 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 trans-fected (pmax dCas9 activators [Tabe 2] + sgRNA expresssion vector or the linear DNA containing the sgRNA expressing fragment produced by PCR), or expressed from a single dual-expression vector [Table 1].
dCas9 activator ORFs (with Stop codon) are also ava-ialble as Gateway donor vectors [Table 3] and can be transferred to Gateway destination vectors (e.g., pmax-DEST [Table 4] and andhttp://www.lifetechnologies.com/us/en/home/life-science/cloning/gateway-cloning/gateway-destination-vectors.html) to adapt to di�erent systems by LR clon-ase reaction.
crispr-on.org
“Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system”
Cell Research doi: 10.1038/cr.2013.122
