Team:Freiburg/Highlights

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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.
...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!