CRISPRa and CRISPRi: Gene Expression Modulation
What is CRISPRa and CRISPRi?
CRISPR/Cas9 has recently adapted to generate two new technologies that modulates gene expression: CRISPRa (CRISPR activation) for gene activation and CRISPRi (CRISPR interference) for gene expression interference. In both CRISPRa and CRISPRi systems, the enzymatically deficient Cas9 (dCas9) is fused or interacts with transcriptional effector(s). dCas9 contains mutations in two active endonuclease domains, losing the capability to cut DNA. However dCas9 can still target a specific DNA location when coupled with a gRNA. In CRISPRa system, dCas9 is fused or interacts with transcriptional activators leading to gene expression upregulation. In CRISPRi, dCas9 is fused with transcriptional repressor(s) leading to gene expression repression. OriGene offers genome wide CRISPRa Kits against all human and mouse genes.
Rules of gRNA design for CRISPRa /CRISPRi
To modulate gene regulation, gRNA is usually designed to target around the promoter region, and the transcriptional start site (TSS) (+1)
OriGene provides fast and cost effective custom gRNA design and cloning service (view details).
Fig. 1. Diagram of CRISPRa and CRISPRi. dCas9 - activator leads to activation; dCas9 - repressor leads to repression.
CRISPRa – CRISPR/Cas9 SAM Synergistic Activation Mediator
CRISPRa SAM is a robust CRISPR gene activation system to activate gene expression using CRISPR technology. The CRISPRa SAM consists of dCas9-VP64, a modified gRNA containing MS2 RNA aptamers, and MS2-p65-HSF1 activation domains. VP64 has four copies of VP16, a viral protein that is used for transcriptional activation. p65 and HSF1 are transcription activation domains. When p65 and HSF1 are brought in close proximity to dCAs9-VP64 via interaction of MS2 with MS2 RNA aptamers in gRNA, the three transactivators can synergistically upregulate gene expression. CRISPRa SAM system can robustly activate both coding and non-coding RNA (lincRNA).
Features:
- Robust CRISPR gene activation system
- Synergistic activation by three activation domains, VP64, p65 and HSF1
- Two vector system, and
- Complement to gene overexpression with cDNA clones
Fig. 2. Diagram of CRISPRa SAM – CRISPR/Cas9 activation system. MS2 in MS2-p65-HSF1 fusion protein binds to MS2 RNA aptamer in the gRNA, bringing p65 and HSF1 to VP64; VP64, p65 and HSF1 then synergistically activate gene expression.
CRISPRa SAM CRISPR/Cas9 Activation Vector Products
| Catalog No. | Product Type | Description |
|---|---|---|
| CRISPRa Vector | pCas-Guide-CRISPRa vector, CRISPRa SAM activation vector containing dCas9-VP64 and gRNA(MS2) cloning site | |
| pCas-Guide-GFP-CRISPRa gene activation vector containing dCas9-VP64 and tGFP reporter gene for gRNA cloning | ||
| pCas-Guide-Puro-CRISPRa Vector, All-in-one Vector for CRISPR/Cas9 Gene activation with Puro selection marker, dCas9-VP64 and gRNA cloning site. | ||
| Enhancer vector | pCRISPRa-Enhancer vector, expressing MS2-p65-HSF1, which synergistically activates gene expression with dCas9-VP64. | |
| Scramble Control | pCas-Guide-CRISPRa-Scramble, scramble gRNA control in GE100055 vector (no selection marker/reporter) | |
| pCas-Guide-GFP-CRISPRa-Scramble, scramble gRNA control in GE100074 vector (with tGFP) | ||
| pCas-Guide-Puro-CRISPRa-Scramble, scramble gRNA control in GE100081 vector (with Puro selection) | ||
| CRISPRa vector Kit | CRISPRa SAM vector kit containing pCas-Guide-CRISPRa (GE100055), pCRISPRa-Enhancer (GE100056) and pCas-Guide-CRISPRa-Scramble (GE100058) |
1. pCas-Guide-CRISPRa Vector
The all-in-one CRISPRa vectors contain both gRNA cloning sites under U6 promoter, and a CMV driven dCas9-VP64 expression cassette. After cloning specific gRNA sequence, the vector can express dCas9-VP64 fusion protein and gRNA with a MS2 binding sequence. Normally the gRNA sequence used in our CRISPRa system is designed based on the promoter sequence of the target gene.
Features:
- Choose from 3 different CRISPRa vectors :
- CRISPRa vector with no selection marker or reporter gene (Cat# )
- CRISPRa vector with tGFP reporter gene (Cat# )
- CRISPRa vector with Puro selection marker (Cat# )
- Express the dCas9-VP64 fusion protein to activate the gene expression at the gRNA targeting site
- Contain the MS2 aptamer in the gRNA loop, which can be used to recruit other regulatory proteins, e.g. the proteins encoded by CRISPRa-Enhancer (Cat# ), and lead to the robust gene activation.
Fig. 3. Schematic diagram of the basic CRISPRa vector (Cat# ). Like other CRISPRa vectors, it encodes dCas9-VP64 fusion protein and gRNA with MS2 aptamer.
pCRISPRa-Enhancer vector encodes a fusion protein of MS2 coat protein and the transactivation domains of p65 and HSF1. MS2 protein will bind to MS2 RNA aptamer in the gRNA, thus bringing p65 and HSF1 together with dCas9-VP64.
Features:
- CMV driven MS2-p65-HSF1
- Requires pCas-Guide-CRISPRa for gene activation
- MS2-p65-HSF1 synergistically activate gene expression with dCas9-VP64
Fig. 4. Schematic diagram of the CRISPRa Enhancer vector (Cat# )
3. Genome-wide predesigned gene activation kit using CRISPRa SAM system
CRISPRa SAM can be used for the up-regulation of gene expression. OriGene offers genome-wide locus specific gene activation kit using CRISPRa SAM system. The specific gRNA is designed to target the promoter region of the gene. It can bring dCas9-VP64 fusion protein to the specific locus, and activate the expression of the target gene.
Each CRISPRa kit contains an enhancer vector, which express a fusion protein of the P65 and HSF1 transactivation domains. The enhancer protein can be recruited to the target gene through the binding of the MS2 sequence in the gRNA loop and enhancer protein, therefore robustly activate gene expression.
Features:
- Turn-key solution for activating endogenous genes, with 3 targeting gRNAs, 1 scramble control, and 1 enhancer vector
- Pre-designed gRNA with OriGene’s proprietary algorithm
- Two vector system, and
- Alternative to gene overexpression with cDNA clones
Fig. 4. Schematic diagram of the CRISPRa Enhancer vector (Cat# )
CRISPRa SAM Validation Data
CRISPRa SAM system robustly activates gene expression
Fig. 5. Target specific pCas-Guide-CRISPRa vector (encodes dCas9-VP64 and gRNA targeting HBG1 or ASCL1 locus) and pCRISPRa-Enhancer (encodes MS2-p65-HSF1) were transfected into HEK293T cells using MegaTran 2.0. Cells were harvested 48 hrs post transfection; qPCR was performed to measure gene expression.
dCas9-VP64 alone can significantly activate gene expression
Fig. 6. dCas9-VP64 and gRNA targeting ASCL1 locus without CRISPRa enhancer was transfected into HEK293T cells using MegaTran 2.0. Cells were harvested 48 hrs post transfection; qPCR was performed to measure gene expression.
CRISPRi – dCas9-KRAB-MeCP2 Gene Interference
| Catalog No. | Product Type | Description |
|---|---|---|
| CRISPRi Vector | pCas-Guide-CRISPRi vector, All-in-one Vector for CRISPR/Cas9 Gene Interference with dCas9-KRAB-MeCP2 and gRNA cloning site. | |
| pCas-Guide-Puro-CRISPRi vector, All-in-one Vector for CRISPR/Cas9 Gene Interference with Puro selection marker, dCas9-KRAB-MeCP2 and gRNA cloning site. | ||
| pCas-Guide-GFP-CRISPRi vector, All-in-one Vector for CRISPR/Cas9 Gene Interference with GFP reporter, dCas9-KRAB-MeCP2 and gRNA cloning site. | ||
| Scramble Control | pCas-Guide-CRISPRi-Scramble, scramble gRNA control in GE100059 vector (no selection marker/reporter) | |
| pCas-Guide-Puro-CRISPRi-Scramble, scramble gRNA control in GE100083 vector (with Puro selection) | ||
| pCas-Guide-GFP-CRISPRi-Scramble, scramble gRNA control in GE100085 vector (with tGFP) |
In this CRISPRi system, dCas9 is fused with KRAB and MeCP2 repression domains to carry out robust gene repression. Krüppel-associated box (KRAB) is a well-known transcriptional repressor domain. MeCP2 has been shown to bind to methylated DNA.
Features:
- All-in-one CRISPRi vector system
- gRNA driven by U6 promoter (after target sequence cloned)
- dCas9-KRAB-MeCP2 fusion protein expression by CMV promoter
- Custom gRNA cloning service into pCas-Guide-CRISPRi
CRISPRi Validation Data
Gene expression repression by dCas9-KRAB-MeCP2
Figure: All-in-one CRISPRi vector containing gene specific gRNA and dCas9-KRAB-MeCP2 was transfected into HEK293T cells Using MegaTran 2.0. gRNA scramble control was used as a negative control. Cells were harvested 48 hrs post transfection and qPCR was performed to measure mRNA expression. Gene expression repression was significant across all genes tested as shown above.