Team:Freiburg/Highlights

6 opportunities with our uniCAS toolkit

We provide 3 different effectors, 2 methods and 1 effector controller! 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, two non-coding RNAs and the appropriate DNA. The 160 kDa large Cas9 protein was mutated by inactivating the DNA cleavage site. The standardization for the iGEM community was done by introducing 10 mutations into the cas9 gene, resulting in the DNA-binding protein dCas9 found in the parts registry. This is the heart of our toolkit: A protein that allows for multiple and sequence-specific DNA binding. By fusing several effector domains to dCas9, we constructed novel engineered proteins for 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 embryonic alkaline phosphatase (SEAP) reporter gene expression. We achieved over 25-fold upregulation of SEAP expression by targeting sequences located upstream of the promoter. The numbers characterizing the crRNAs in the graph represent the distance from the translation start site. Read more!

Epigenetic Repression

Specific chromatin modification was achieved by fusing the histone methyltransferase G9a to dCas9 and 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 VEGF-8 corresponds to a region in the VEGF locus at position -8 bp from the transcription start site (TSS) and VEGF-573 corresponds to a region -573 bp from the 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 when targeting different DNA sites. The numbers characterizing the crRNAs represent the distance from the transcription start site. Read more!

Light control

We aimed to control our system with light thereby allowing for gene regulation with high spatiotemporal resolution. We engineered a system for induction by red, UVB and blue light. The blue light system is based on the light-triggered interaction of CRY2 and CIB1. CRY2 was fused to dCas9 which can upon light-stimulus recruit the CIB1-VP16 fusion protein to any DNA sequence of interest. A 5-fold upregulation of SEAP reporter was achieved (*, p<0.05). dCas9-CRY2 was targeted to four DNA regions upstream of the promoter. The numbers characterizing the crRNAs represent the distance from the translation start site. 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 easily 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. Gene regulation worked even more efficiently when using multiple targets. Read more!

Manual

As we believe that our engineered CRISPR/Cas9 system is a promising tool for targeted gene regulation, we would like to offer a manual to the iGEM community for facilitated usage of our toolkit. Therefore we designed an interactive Manual Tool that generates detailed descriptions for your own gene regulation experiments dependent on whether you would like to effciently repress or activate gene expression. We provide all our experimental knowledge and optimized protocols to everyone who would like to use our uniCAS toolkit. Read more!

uniCAS Binding Assay - uniBAss

We developed a novel and innovative ELISA-based method to quantify the binding efficiencies of our dCas9 fusion proteins: The uniCAS Binding Assay (uniBAss). Therefore, biotinylated oligos were coated on 96-well ELISA plates via the interaction with streptavidin. We were able to show that it is a powerful tool for characterizing the modified dCas9 fusion proteins by assessing their DNA binding capacity with possible improvements for high-throughput screenings. Read more!

To summarize - In the last months we were able to ...

• ... construct a catalytically inactive version of Cas9 (dCas9) and thus generate a DNA binding protein.
• ... make our dCas9 accessible to the whole iGEM community by mutating illegal iGEM restriction sites.
• ... combine this modified dCas9 with different transcriptional effectors.
• ... express these fusion proteins in various mammalian cell lines.
• ... control mammalian gene expression via our modified CRISPR/Cas9 fusion proteins.
• ... build devices for controlling gene expression by different light stimuli.
• ... provide an RNA plasmid termed RNAimer for facile insertion of crRNAs which target desired target sequences.
• ... build an online tool that generates customized manuals for easy usage of our toolkit.
• ... develop a method to assess the DNA binding capacity of our dCas9 fusion proteins - the uniBAss.
• ... model our system and obtain a possible insight into behavior of for measured and unmeasured components.

... design, develop, test and now offer a universally applicable toolkit for efficient gene
regulation to the synthetic biology community.