Team:Freiburg/Highlights

9 opportunities of our uniCAS toolkit

We provide 3 different effectors to efficiently activate or repress genes in mammalian cells. Our toolkit also comprises devices for controlling effectors by light stimuli. We furthermore constructed a light box called uniBOX to conduct light experiments. 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. With our modeling data we concluded how to optimize our system - by truncating dCas9.

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 Cas9 protein was mutated to inactivate 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.

Multiple Targeting

One of the greatest advantages of the CRISPR/Cas9 system is that only one protein is necessary 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!

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 an over 25-fold upregulation of SEAP expression by targeting sequences located upstream of the promoter. 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 (*, p<0.05) 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 to allow 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 - upon light-stimulus - can 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 simultaneously to four DNA sites upstream of the promoter. Numbers characterizing the crRNAs represent the distance from the translation start site. Read more!

uniBOX - A LEGO built light box

Light inducible systems are more and more used in iGEM and synthetic biology in general. Our gene regulation system is controllable via light as well. Therefore we thought of an easy and affordable way to build a light box to illuminate our cells with a certain wavelength. But construction of such a light box can be hard because there are certain problems you have to face. Our so called uniBOX can be built by using common things like LEGO bricks, glass, foil and LEDs. We were able to show that light systems can be controlled efficiently and we now provide a do-it-yourself manual for building your own uniBOX! Read more and take a look at our manual!

Modeling

We used a kinetic approach to model and characterize our system. It is based on various ordinary differential equations which describe the behaviour of our network. To optimize our system we varied dCas9 production rates and noticed that especially a higher expression rate of dCas9 was able to increase the efficiency of the system. Since dCas9 is a large protein, consisting of 160 kDa, expression rate of a smaller truncated version would be higher. See the next slide for truncation ideas and results. Read more about our modeling!

Truncation

To increase the efficiency of our system we truncated the dCas9 protein at different sites and various extent. Expression rates of smaller proteins were higher as showed in the western blots. In accordance with our modeling this should lead to a higher rate of gene activation or repression. However our functional tests of the truncated proteins were yet inconclusive. 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). Biotinylated oligos are coated on 96-well ELISA plates via the interaction with streptavidin and bound dCas9/RNA complexes can be detected via antibodies. We were able to show that this is a powerful tool to characterize the modified dCas9 fusion proteins by assessing their DNA binding capacity with possible improvements for high-throughput screenings. 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 efficiently 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!

Application

Our uniCAS toolkit is a versatile applicable tool. Every expert we talked to had amazing ideas concerning application in their own research!

“… the use of this toolkit may help us in understanding complex metabolomes, because many enzymes can be regulated at once …” - Prof. Dr. Hess, Genetics of Cyanobacteria

“... I would use this kit to understand the transcriptional codes of brain development better, by simultaneously regulating various promoters of neural transcription factors ...“ - Dr. Jochen Holzschuh, Neuronal Development

Read more!

Human Practice & Outreach

In case of safety and ethical issues concerning our project we consulted many biology experts and asked for their opinion and advice. Next we connected synthetic biology with art and music as all the three disciplines require creativity, technical skills and a lot of commitment and also art & music can be used to address a lot of people. We further organized several events to inform about synthetic biology and our project. We went to a political party and schools, participated at an ethical seminar, a science fair and collaborated with other German teams to organize the SynBio Day. 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 10 illegal iGEM restriction sites.
• combine this modified dCas9 with different transcriptional effectors and express them in 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 DNA sequences.
• create 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 measured and unmeasured components, which led us to conclusions how our system could be optimized.
• truncate dCas9 in order to increase expression rate and express these protein versions.
• build a cheap and functioning light box, called "uniBOX", for light experiments.

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