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. pii: S0092-8674(13)00826-X. doi: 10.1016/j.cell.2013.06.044.
(Link opens in a new window)
PubMed
(Link opens in a new window)
Article
The CRISPR ( Clustered Regularly Interspaced Short Palindromic Repeats) system offers a general approach for RNA-guided regulation of transcription. We have shown that fusion of CRISPR-associated catalytically inactive dCas9 protein to distinct effector domains (e.g. VP64, p65AD, KRAB, and Mxi1) enables robust and efficient repression or activation of transcription in human and yeast cells, with the site of delivery determined solely by a co-expressed short guide RNA (sgRNA). Coupling of dCas9 to a transcriptional repressor domain can effectively silence expression of multiple endogenous genes, with no detectable off-targets as verified by RNA-seq analysis. Here, we provide a modular and general CRISPR toolbox for the precise regulation of gene expression in eukaryotic cells.
1. CRISPRi transcription regulation system in human cells
The dCas9 fusion plasmids contain a human codon optimized dCas9 gene that is fused to different effector domains, under the control of either a spleen focus-forming virus (SFFV) or a murine Stem cell retrovirus LTR promoter. The sgRNA plasmids contain a murine U6 promoter controlled sgRNA cassette, wherein the GN19 can be custom designed to target sequences in the genome. The sgRNA plasmid also contains a CMV-puro-t2A-mCherry expression cassette, for selection or fluorescent gating of transiently transfected cells.
2. CRISPRi transcription regulation system in yeast
The dCas9 fusion CEN/ARS plasmids contain a human codon optimized dCas9 fused with two C-terminal SV40 nuclear localization signal sequences and an Mxi1 repressive domain. The sgRNA CEN/ARS plasmids contain a SNR52 promoter, sgRNA, and SUP4 terminator 3’ flanking sequence.
Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 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 |
| 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) |
| 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) |
| 46917 | pU6-sgCXCR4-2 | Human pSico-based U6 vector containing murine U6 promoter and sgRNA targeting endogenous CXCR4 gene |
| 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 |
| 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 NLS and Mxi1 domain controlled by TDH3 promoter |
| 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 |