Team:Freiburg/Highlights

6 opportunities with our uniCAS toolkit

We provide 3 different effectors, 2 methods & 1 device for effector control! By using our toolkit it is possible to efficiently activate or repress genes in mammalian cells. Furthermore, our toolkit comprises devices for controlling effectors by light stimuli. Use our custom-tailored Manual Tool to generate detailed instructions for your own CRISPR/Cas9 based-gene regulation experiment. With our toolkit and the standardized RNA-plasmid termed RNAimer it is possible to target not only one, but multiple genes of interest. We also developed uniBAss - our universal binding assay for assessing the binding capacity of our fusion proteins.

dCas9 - The heart of our toolkit

The CRISPR/Cas9 system relies on a protein-RNA-DNA interaction between the Cas9 protein and two non-coding RNAs. The 160 kDa large Cas9 protein was mutated by inactivating the DNA cleavage site and standardized for the iGEM community by introducing 10 mutations into the cas9 gene resulting in the DNA-binding protein dCas9 found in the parts registry (BBa_K1150000).

This is the heart of our toolkit: A protein that allows multiple and sequence-specific DNA binding. By fusing various effector domains to dCas9, we constructed novel proteins allowing efficient gene regulation. Read more in the next slides.

Activation

We fused dCas9 to the trans-activation domain VP16. This fusion protein is able to activate gene expression from a minimal CMV promoter. The fusion protein was successfully tested in mammalian cells and used to activate the secreted alkaline phosphatase (SEAP) reporter gene expression. We achieved up to 30-fold upregulation of SEAP expression by targeting sequences that are upstream of the promoter in which target 1 equals a sequence in the ß-lactamase gene and target 2 represents the EMX1 sequence. Read more!

Chromatin modification (Repression)

Specific chromatin modification was achieved by fusing the histone methyltransferase G9a to dCas9 thereby contributing an epigenetic BioBrick. G9a primarily methylates Histone H3. Different endogenous vegf loci were targeted in mammalian cells. This resulted in an up to 50 % repression in which target 3 corresponds to a region in the vegf loci at position -8 bp from the transcription start site (TSS) and target 4 equals vegf -573 from ths TSS. Read more!

Repression

The transcriptional repressor domain Krüppel associated box (KRAB) was fused to dCas9. Thus, a transcriptional repressor with the flexibility to target any DNA sequence of interest was engineered. The device was tested in mammalian cells to target endogenous vegf loci. An up to 50 % repression was achieved in which target 4 corresponds to a region in the vegf loci at position -573 bp from the TSS and target 5 equals to vegf position +343 from the TSS. Read more!

Multiple Targeting

One of the greatest advantages of the CRISPR/Cas9 system is that only one protein is required for targeting of various DNA sequences. The only component which needs to be replaced is the CRISPR-RNA (crRNA). We therefore designed an RNA plasmid termed the RNAimer. It provides the backbone for easiy exchanging the sequence for these crRNAs. Functional tests showed that the RNAimer plasmid works efficiently in mammalian cells. For multiple targeting, different crRNAs can be combined into one RNAimer plasmid. We could show that gene regulation worked even more efficiently when using multiple targets Read more!.

Manual

We wanted not only to share our BioBricks, but also our experience. Therefore we desinged an interactive Manual Tool, that generates personalized descriptions for gene regulation experiments. We provide all our experimental knowledge and optimized protocols to everyone who wants to use our uniCAS toolkit. Read more!https://2013.igem.org/Team:Freiburg/parts/sharing

uniCas binding assay - uniBAss

We developed a novel and innovative ELISA-based method for quantifying the binding efficiencies of our dCas9 proteins: The uniCAS binding assay uniBAss. Biotinylated oligos are coated on 96-well plates via the interaction with strepatvidin. It could be shown that it is a powerful tool for characterizing the modified dCas9 fusion proteins by assessing its DNA binding capacity with possible improvements for high-throughput screenings.Read more!https://2013.igem.org/Team:Freiburg/Project/method

To summarize - 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 "RNAimer" 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.