CRISPR Activation (CRISPRa) Service


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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.

3 common CRISPRa technologiesFigure.1 Common CRISPRa technologies. (Chavez et al. 2016)

Among the many designs, the three technologies of VPR, SAM and Suntag are the most popular:

The three functional components 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 relevant technologies and experience. 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 2), which can really help our customers achieve gene transcription activation.

Three CRISPRa technologies have excellent transcriptional activation ability and specificity
Figure.2 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. Chavez A, et al. (2015) Highly efficient Cas9-mediated transcriptional programming. Nature methods 12:326-328.
  2. Konermann S, et al. (2015) Genome-scale transcriptional activation by anengineered CRISPR-Cas9 complex. Nature 517:583-588.
  3. Tanenbaum ME, et al. (2014) A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159:635-646.
  4. Chavez A, et al. (2016). Comparison of Cas9 activators inmultiple species. Nature methods 13:563-567.
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