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Team:Freiburg/Highlights
From 2013.igem.org
- ...to design a catalytically inactive version of Cas9 and designing a new class of DNA binding proteins.
- ...to combine this modified Cas9 with different effectors.
- ...to express the system in various mammalian cell lines.
- ...to control human gene expression via our modified CRISPR/Cas system.
- ...to control gene expression on light stimulus.
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 Cas9 we altered the properties it in various ways.
The activation domain VP16 is able to activate transcription of genes.
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| Figure 1: Bildunterüberschrift HIER DANN ALLES ANDERE |
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| Figure 1: Bildunterüberschrift HIER DANN ALLES ANDERE |
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| Figure 1: Bildunterüberschrift HIER DANN ALLES ANDERE |
We were able to induce our system on light stimulus. This was possible by using photorecetors of higher plants.
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.
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 Cas9 and the locus specific RNA.
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| Figure 1: Bildunterüberschrift HIER DANN ALLES ANDERE |
In summary, we established a new modularized tool kit for modulating gene expression: The uniCAS Toolkit!
