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Team:Freiburg/Highlights
From 2013.igem.org
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<ul style="font-size:20px"> | <ul style="font-size:20px"> | ||
| - | <li>... | + | <li>...constuct a <b>catalytically inactive version of Cas9</b> and this way generate a new class of DNA binding proteins.</li> |
| - | <li>...combine this modified dCas9 with <b>different effectors</b>.</li> | + | <li>...combine this modified dCas9 with <b>different transcriptional effectors</b>.</li> |
| - | <li>...express | + | <li>...express this system in various <b>mammalian cell lines</b>.</li> |
<li>...control human <b>gene expression</b> via our modified CRISPR/Cas system.</li> | <li>...control human <b>gene expression</b> via our modified CRISPR/Cas system.</li> | ||
<li>...control gene expression on <b>light stimulus</b>.</li> | <li>...control gene expression on <b>light stimulus</b>.</li> | ||
| - | <li>...<b> | + | <li>...make our <b>dCas9 accesible to the whole iGEM comunity</b> by mutating illegal iGEM restriction sites</b>.</li> |
<br> | <br> | ||
In summary we build up a <b>universal toolkit for gene regulation</b>. | In summary we build up a <b>universal toolkit for gene regulation</b>. | ||
Revision as of 20:26, 30 September 2013
Highlights
In the last months we were able to...
- ...constuct a catalytically inactive version of Cas9 and this way generate a new class of DNA binding proteins.
- ...combine this modified dCas9 with different transcriptional effectors.
- ...express this system in various mammalian cell lines.
- ...control human gene expression via our modified CRISPR/Cas system.
- ...control gene expression on light stimulus.
- ...make our dCas9 accesible to the whole iGEM comunity by mutating illegal iGEM restriction sites.
In summary we build up a universal toolkit for gene regulation.
In the beginning
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.
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| 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
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| Figure 2: Repression via dCas9:KRAB Using dCas9:KRAB we were able to repress GFP expression in mammalian cells. |
Chromatin modification (Repression)
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| 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 photoreceptors 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.
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| Figure 4: uniBAss We developed an assay for testing the binding capacity of our constructs. |
Conclusion
We established a new modularized toolkit for modulating gene expression specifically: The uniCAS Toolkit!
