"
Team:Freiburg/Highlights
From 2013.igem.org
Highlights
In the last months we were able to...
- ...design a catalytically inactive version of Cas9 and design a new class of DNA binding proteins.
- ...combine this modified dCas9 with different effectors.
- ...express the system in various mammalian cell lines.
- ...control human gene expression via our modified CRISPR/Cas system.
- ...control gene expression on light stimulus.
- ...standardize dCas9 by mutating illegal iGEM restriction sites.
Our toolkit ...
A mutated Cas9 derived protein without nickase function was our start. This is basically a DNA binding protein, that is relying on a protein-RNA-DNA interaction.
By fusing effector domains to dCas9 we altered the properties in various ways.
Activation
The activation domain VP16 is able to activate transcription of genes.
 |
| Figure 1: Activation by Cas9:VP16 By fusing the transcriptional activation domain VP16 to dCas9, we are able to activate a SEAP reporter transcription. |
Repression
The fusion of the transcriptional repressor domain KRAB leads to synthetic repression of gene expression.  |
| Figure 2: Repression via dCas9:KRAB Using dCas9:KRAB we were able to repress GFP expression in mammalian cells. |
Chromatin modification (Repression)
Specific chromatin modification was achieved by fusing a histone methyltransferase G9a to dCas9. With this protein we are able to specifically repress endogenous gene expression.
 |
| Figure 3: Endogenous, stable repression by dCas9:G9a Chromatin remodeling, resulting in repression of endogenous genes is possible by fusing the histone methyltransferase G9a to dCas9. |
Light switch
We were able to induce our system on light stimulus. This was possible by using photorecetors of higher plants.
Targeting with RNAimer
By building a plasmid containing the necessary RNAs and insertion sites for targeting we created a modular, BioBrick compatible system for multiple DNA targeting: The RNAimer. Using our RNAimer plasmid it is easy to combine several target sequences on one plasmid using the BioBrick standard.
uniBAss - Binding Assay
We developed an ELISA based method. With this method we can quantify the binding efficiency of our proteins. We called this binding assay uniBAss. It is a powerful tool for the characterization of the interaction between the modified dCas9 and the locus specific RNA.
 |
| Figure 4: uniBAss We developed an assay for testing the binding capacity of our constructs. |
Conclusion
In summary, we established a new modularized toolkit for modulating gene expression: The uniCAS Toolkit!
