Qi Labs CRISPR Plasmids Available from Addgene
We have recently repurposed the bacterial immune system, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) 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 single guide 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 PMID 23452860. PMC3664290.
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 PMID 23849981.
Individual plasmids can be ordered via the links below:
|44246||pdCas9-humanized||A catalytically inactive, human codon-optimized Cas9 expression plasmid|
|44247||pdCas9::BFP-humanized||A catalytically inactive, human codon-optimized Cas9-BFP fusion expression plasmid|
|44248||pgRNA-humanized||A customizable gRNA expression plasmid for use with either human codon-optimized Cas9 constructs|
|44249||pdCas9-bacteria||A catalytically inactive bacterial Cas9 expression plasmid|
|44250||pwtCas9-bacteria||A wild-type Cas9 expression plasmid for use editing bacterial genomes|
|44251||pgRNA-bacteria||A customizable gRNA expression plasmid for use with the bacterial Cas9 constructs|
|46910||pHR-SFFV-dCas9-BFP||Human expression vector containing SFFV promoter, dCas9 that is fused to 2x NLS and tagBFP|
|46911||pHR-SFFV-dCas9-BFP-KRAB||Human expression vector containing SFFV promoter, dCas9 that is fused to 2x NLS, tagBFP and a KRAB domain|
|46912||pMSCV-LTR-dCas9-VP64-BFP||Human expression vector containing MSCV LTR promoter, dCas9 that is fused to 2x NLS, VP64 and tagBFP|
|51023||pSLQ1658-dCas9-EGFP||Human expression vector containing dCas9 that is fused to 2x NLS and EGFP for CRISPR imaging|
|51024||pSLQ1651-sgTelomere(F+E)||Lentiviral vector with sgRNA targeting human telomeres|
|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|
|46914||pU6-sgGFP-NT1||Human pSico-based U6 vector containing murine U6 promoter and sgRNA targeting GFP (NT1)|
|46917||pU6-sgCXCR4-2||Human pSico-based U6 vector containing murine U6 promoter and sgRNA targeting endogenous CXCR4 gene|
|46920||pTDH3-dCas9||Yeast CEN/ARS vector (Leu2) that contains dCas9 fused to NLS controlled by TDH3 promoter|
|46921||pTDH3-dCas9-Mxi1||Yeast CEN/ARS vector (Leu2) that contains dCas9 fused to N|
|51025||pSLQ1661-sgMUC4-E3(F+E)||Lentiviral vector that contains sgRNA targeting repetitive sequence of human MUC4 exon 3|
|46915||pU6-sgGAL4-1||Human pSico-based U6 vector containing murine U6 promoter and sgRNA targeting GAL4 UAS promoter|
|46916||pU6-sgGAL4-4||Human pSico-based U6 vector containing murine U6 promoter and sgRNA targeting GAL4 UAS promoter (negative control)|
|46918||pU6-sgCD71-2||Human pSico-based U6 vector containing murine U6 promoter and sgRNA targeting endogenous CD71 gene|
|46919||pMLS-SV40-EGFP||Target EGFP gene that is stably integrated into HEK293 genome|
|46922||pSNR52-sgTEF1||Yeast CEN/ARS vector (Ura3) that contains sgRNA controlled by SNR 52 promoter, targeting endogenous TEF1 promoter|
|46923||pSNR52-sgTET||Yeast CEN/ARS vector (Ura3) that contains sgRNA controlled by SNR 52 promoter, targeting endogenous TRE elements of pTET07 promoter|