Qi Labs CRISPR Plasmids Available from Addgene
We have recently repurposed the bacterial immune system, CRISPR ( C lustered R egularly I nterspaced S hort P alindromic R epeats) pathway, as an RNA-guided DNA binding platform, to repress expression of arbitrary genes in bacteria or human cells ( Qi et al. ). This CRISPR interfering system (CRISPRi), works independently of host cellular machineries, requiring only a nuclease-deficient Cas9 (dCas9) protein and a customized s ingle g uide RNA (sgRNA) designed with a 20-basepair complementary region to any gene of interest. Co-expression of dCas9 and sgRNA can efficiently block transcription (in bacteria, ~300-fold repression) by interfering with transcriptional elongation, RNA polymerase binding, or transcription factor binding.
The binding specificity is determined jointly by 20-bp matching region on the sgRNA and a short DNA motif (protospacer adjacent motif or PAM, sequence: NGG) juxtaposed to the DNA complementary region. The uniqueness of CRISPRi, as compared to several recently published works on applying the wild-type CRISPR system for genome mutagenesis ( Cong et al. , Mali et al. , Jiang et al. , Hwang et al. ), is that the nuclease-deficient mutant could silence transcription on the gene expression level without genetically altering the target loci. Thus, CRISPRi is a system that can regulate a genome instead of modifying a genome.
1. Two-plasmid CRISPRi system for bacterial gene knockdown
The first plasmid ( pdCas9_bacteria ) contains an anhydrotetracycline (aTc)-inducible dcas9 gene on a p15A vector with chloramphenicol resistance. The second plasmid ( pgRNA_bacteria ) contains a constitutive sgRNA expression cassette on a ColE1 vector with ampicillin resistance, wherein the N20 can be custom designed to target arbitrary sequences in the genome. Co-expression of both plasmids in bacteria could cause up to 300-fold repress on targeted genes.
2. Two-plasmid CRISPRi system for mammalian gene knockdown
The first plasmid ( pdCas9_humanized ) contains a human codon optimized dcas9 gene under the control of Murine Stem Cell retroVirus LTR promoter. The second plasmid ( pgRNA_humanized ) contains a murine U6 promoter controlled sgRNA cassette, wherein the GN19 can be custom designed to target sequences in the genome. The pgRNA_humanized also contains a CMV-puro-t2A-mCherry expression cassette, useful for selection or fluorescent gating of transiently transfected cells. Co-expression of both plasmids in HEK293 cells could cause up to 2~3-fold repress on targeted fluorescent genes.
These plasmids are described in:
Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Cell . 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022. PubMed . PMC .
CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS. Cell . 2013 Jul 9. doi: 10.1016/j.cell.2013.06.044. PubMed .
Individual plasmids can be ordered via the links below:
|44246||pdCas9-humanized||A catalytically inactive, human codon-optimized Cas9 expression plasmid||Add to Cart|
|44247||pdCas9::BFP-humanized||A catalytically inactive, human codon-optimized Cas9-BFP fusion expression plasmid||Add to Cart|
|44248||pgRNA-humanized||A customizable gRNA expression plasmid for use with either human codon-optimized Cas9 constructs||Add to Cart|
|44249||pdCas9-bacteria||A catalytically inactive bacterial Cas9 expression plasmid||Add to Cart|
|44250||pwtCas9-bacteria||A wild-type Cas9 expression plasmid for use editing bacterial genomes||Add to Cart|
|44251||pgRNA-bacteria||A customizable gRNA expression plasmid for use with the bacterial Cas9 constructs||Add to Cart|
|46910||pHR-SFFV-dCas9-BFP||Human expression vector containing SFFV promoter, dCas9 that is fused to 2x NLS and tagBFP||Add to Cart|
|46911||pHR-SFFV-dCas9-BFP-KRAB||Human expression vector containing SFFV promoter, dCas9 that is fused to 2x NLS, tagBFP and a KRAB domain||Add to Cart|
|46912||pMSCV-LTR-dCas9-VP64-BFP||Human expression vector containing MSCV LTR promoter, dCas9 that is fused to 2x NLS, VP64 and tagBFP||Add to Cart|
|51023||pSLQ1658-dCas9-EGFP||Human expression vector containing dCas9 that is fused to 2x NLS and EGFP for CRISPR imaging||Add to Cart|
|51024||pSLQ1651-sgTelomere(F+E)||Lentiviral vector with sgRNA targeting human telomeres||Add to Cart|
|46913||pMSCV-LTR-dCas9-p65AD-BFP||Human expression vector containing MSCV LTR promoter, dCas9 that is fused to 2x NLS, p65 activation domain and tagBFP||Add to Cart|
|46914||pU6-sgGFP-NT1||Human pSico-based U6 vector containing murine U6 promoter and sgRNA targeting GFP (NT1)||Add to Cart|
|46917||pU6-sgCXCR4-2||Human pSico-based U6 vector containing murine U6 promoter and sgRNA targeting endogenous CXCR4 gene||Add to Cart|
|46920||pTDH3-dCas9||Yeast CEN/ARS vector (Leu2) that contains dCas9 fused to NLS controlled by TDH3 promoter||Add to Cart|
|46921||pTDH3-dCas9-Mxi1||Yeast CEN/ARS vector (Leu2) that contains dCas9 fused to NLS and Mxi1 domain controlled by TDH3 promoter||Add to Cart|
|51025||pSLQ1661-sgMUC4-E3(F+E)||Lentiviral vector that contains sgRNA targeting repetitive sequence of human MUC4 exon 3||Add to Cart|
|46915||pU6-sgGAL4-1||Human pSico-based U6 vector containing murine U6 promoter and sgRNA targeting GAL4 UAS promoter||Add to Cart|
|46916||pU6-sgGAL4-4||Human pSico-based U6 vector containing murine U6 promoter and sgRNA targeting GAL4 UAS promoter (negative control)||Add to Cart|
|46918||pU6-sgCD71-2||Human pSico-based U6 vector containing murine U6 promoter and sgRNA targeting endogenous CD71 gene||Add to Cart|
|46919||pMLS-SV40-EGFP||Target EGFP gene that is stably integrated into HEK293 genome||Add to Cart|
|46922||pSNR52-sgTEF1||Yeast CEN/ARS vector (Ura3) that contains sgRNA controlled by SNR 52 promoter, targeting endogenous TEF1 promoter||Add to Cart|
|46923||pSNR52-sgTET||Yeast CEN/ARS vector (Ura3) that contains sgRNA controlled by SNR 52 promoter, targeting endogenous TRE elements of pTET07 promoter||Add to Cart|