CRISPR Activation (CRISPRa) Service

CRISPR Activation (CRISPRa) Service Inquiry

Before the birth of CRISPR technology, there are no suitable technology to activate endogenous gene expression for biological community. The Tetracycline (Tet)-on/off system and GAL4-UAS system both induce ectopic expression. Besides, directly transfected expression vectors for gene overexpression and screening cDNA libraries for gene overexpression are also routine methods. But no matter which method, it is necessary to obtain the cDNA sequence of the gene in advance, and the construction cost is relatively high, especially for large-scale cDNA library, which is very difficult to produce.

The emergence of CRISPR technology allows us to activate gene expression in situ. The earliest CRISPRa system used the same VP16 domain as the Tet-on/off system to activate gene transcription. However, unlike the artificially designed TRE promoter, the endogenous gene promoter is subject to complex regulation. Even with 10 VP16 copies of VP160, the ability of CRISPRa to activate endogenous genes is still limited. When endogenous genes are transcribed, they usually recruit many proteins to form a huge complex. Based on the endogenous transcription process, researchers begin to use the powerful transformation capabilities of Cas9 and sgRNA to fuse or recruit multiple proteins, so as to enhance the purpose of transcription.

CRISPR Activation (CRISPRa) 2.0

Through the modification of the CRISPR/dCas9 system, especially the modification of the guide RNA, the dCAS9 protein and the guide RNA can recognize the protein at the same time, and adsorb more transcription activators, thereby achieving a stronger transcription activation effect. The first generation of gene activation technology is about 7 times, while the second generation of gene activation technology can be as high as 40 times.

The activation mechanism of a eukaryote-like long distance regulation in CRISPRa 2.0 design.
Figure 1. The activation mechanism of a eukaryote-like long distance regulation in CRISPRa 2.0 design. (Yang L, et al. 2019)

CRISPR Activation (CRISPRa) 3.0

The third-generation gene activation technology mainly relies on the combination of multi-gene targeting technology and gene activation technology. By designing multiple guide RNAs, this multi-gene activation technology can act on all related genes in the same metabolic pathway, thereby achieving an increase in the overall pathway expression. This gene activation technology is very effective in dicotyledonous plants, By activating the flowering genes of Arabidopsis thaliana, the flowering time of Arabidopsis can be shortened from 40 days to 13 days, realizing strong activation of biallelic genes.

CRISPR Activation (CRISPRa) Structural Design

Among the many designs, the three technologies VPR, SAM and Suntag are the most popular ones (Figure 1):

3 common CRISPRa technologies
Figure 2. Common CRISPRa technologies. (Chavez et al. 2016)

The three functional components (VP64, p65, RTA) in VPR technology play a synergistic role, enhancing the ability of the fusion protein to activate gene transcription (Chavez et al. 2015).

SAM (synergistic activation mediator) technology utilizes the ability of sgRNA to transform, adding MS2 sequence to the two neck-loop structures of sgRNA to recruit MCP protein, and MCP protein is fused with P65 and hsf1 domains. As a result, the fusion of VP64 domains on dCas9 is synergistic, and more activation originals have a stronger activation effect (Konermann    et al. 2015).

Suntag technology uses the principle of antigen and antibody. The long-chain antigen fused to dCas9 can recruit VP64 carried by multiple antibody scFv at a time, amplifying its activation effect (Tanenbaum et al. 2014).

Lifeasible has been deeply involved in the field of gene editing for several years, and has accumulated a wealth of experience for relevant technologies. We are devoted to optimizing the CRISPR system to carry more activation elements. After synergistic amplification, the CRISPRa system has a stronger activation effect. Their final effect is significantly better than the earliest used VP64, and there is no obvious off-target phenomenon (Figure 3), which can really help our customers achieve gene transcription activation.

Three CRISPRa technologies have excellent transcriptional activation ability and specificity
Figure 3. Three CRISPRa technologies have excellent transcriptional activation ability and specificity (Chavez et al. 2016)

CRISPRa experiment procedure

CRISPRa experiment procedure

We hope to share our technology and results with customers to help customers advance their projects better and faster. For inquiries, please contact Lifeasible.

References

  1. Liu Y, et al. (2019) Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria. Nature Communications 10(1):1-16.
  2. Chavez A, et al. (2015) Highly efficient Cas9-mediated transcriptional programming. Nature methods 12:326-328.
  3. Konermann S, et al. (2015) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517:583-588.
  4. Tanenbaum ME, et al. (2014) A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159:635-646.
  5. Chavez A, et al. (2016). Comparison of Cas9 activators in multiple species. Nature methods 13:563-567.
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