Team:Freiburg/Highlights

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<td> <b>Figure 1: Bildunterüberschrift </b><br>
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<td> <b>Figure 1: Activation by Cas9:VP16 </b><br>
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HIER DANN ALLES ANDERE
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By fusing the transcriptional activation domain VP16 to Cas9, we are able to activatea SEAP reporter transcription.
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Revision as of 15:34, 29 September 2013

Highlights

In the last months we were able...

  • ...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.

Figure 1: Activation by Cas9:VP16
By fusing the transcriptional activation domain VP16 to Cas9, we are able to activatea SEAP reporter transcription.
The fusion of the transcriptional repressor domain KRAB leads to synthetic repressor of gene expression.

Figure 1: Bildunterüberschrift
HIER DANN ALLES ANDERE
Specific chromatin modification was achieved by fusing a histone methyl transferase G9a to dCas9. With this protein we are able to specifically repress endogenous gene expression.

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.

Figure 1: Bildunterüberschrift
HIER DANN ALLES ANDERE

In summary, we established a new modularized tool kit for modulating gene expression: The uniCAS Toolkit!

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