Team:Freiburg/Highlights/2

In the last months we were able to ...

• ... construct a catalytically inactive version of Cas9 and thus generate a DNA binding protein.
• ... combine this modified dCas9 with different transcriptional effectors.
• ... express this fusion proteins in various mammalian cell lines.
• ... control mammalian gene expression via our modified CRISPR/Cas fusion proteins.
• ... build devices for controling gene expression by light stimulus.
• ... provide an RNA plasmid for easily inserting sequences for crRNAs which target every desired target.
• ... build an online tool that generates customized manuals for using our toolkit
• ... develop a method to assess the DNA binding capacity of our dCas9-fusion proteins.
• ... make our dCas9 accessible to the whole iGEM community by mutating illegal iGEM restriction sites.
• ... In summary, we can now offer a universally applicable toolkit for gene regulation.

6 opportunities with our uniCAS toolkit

We provide 3 different effectors, 2 methods & 1 effector controller! Using our toolkit it's possible to efficiently activate or repress genes. We also provide devices for effector controling by light. Use our custom-tailored Manual Tool to generate your individual manual for your needs of gene regulation. Further it's possible to target not only one, but multiple genes of interest! And we established uniBAss - our universal Binding Assay. Best of all: It's open source and in iGEM standard!

dCas9 - The Heart of our toolkit

We started by mutating the DNA cleavage site in the Cas9 protein and generated a sequence specific DNA binding protein that is relying on a protein-RNA-DNA interaction. We are now able to influence the DNA binding locus and can direct the protein to requested DNA targets.
This is the heart of our toolkit. A protein that allows multiple and sequence specifid DNA binding.

For simple and flexible regulation we do need now effectors, that can be fused to the protein.

Activation

For activation we choosed VP-16 as effector. Through the transactivating function of VP-16 the expression of these genes will be enhanced.
We achieved up to 30-fold activation.

Repression

The fusion of the transcriptional repressor domain KRAB leads to synthetic repression of gene expression. With this construct a strong repression could be observed.

Chromatin modification (Repression)

Specific chromatin modification was achieved by fusing the histone methyltransferase G9a to dCas9. With this protein we are able to specifically repress endogenous gene expression.

Multiple Targeting

One of the biggest advantages of the CRISPR/Cas9 system is that only one protein is required for targeting several DNA sites: For a new target there has to be just another guiding RNA. We designed an RNA plasmid, "RNAimer", containing this RNA. For multiple targeting different RNAimers can be easily combined using the iGEM BioBrick system.

And as the results show, multiple targeting is possible and even better!

uniBAss

We developed a novel and innovative ELISA based method to quantify the binding efficiency of our proteins. We called this binding assay uniBAss. This is a powerful tool for characterizing the modified dCas9 by assessing its DNA binding capacity with high throughput capabilities.

Figure 5: uniBAss - universal Binding Assay First step ...