Team:Freiburg/Project/1

Introduction

Our Team developed a universal toolkit, termed uniCAS, that enables customizable gene regulation in mammalian cells. Therefore, we engineered the recently understood and highly promising CRISPR/Cas9 system. The regulation is based on the RNA-guided Cas9 protein, which allows specific targeting of DNA sequences. Our toolkit comprises not only a standardized Cas9 protein, but also different effector domains for efficient gene activation or repression. We further engineered a modular RNA plasmid for easy implementation of RNA guide sequences. As an additional feature, we established an innovative screening method for assessing the functionality of our uniCAS fusion proteins. Single genes and even whole genetic networks can be modified using our uniCAS toolkit. We think that our toolbox of standardized parts of the CRISPR/Cas9 system offers broad application in research fields such as tissue engineering, stem cell reprogramming and fundamental research.

Introduction to our project

The enormous amount of gene regulation, signal transduction and metabolic pathways gives us a slight idea about what complexity of life really means. This astonishing complexity is one of the most crucial things to understand, if we want to understand life itself.

Many approaches have been investigated and given us insights into these biological pathways, resulting in more and more elaborate tools. Solutions for the treatment of systemic diseases like cancer or the fine-tuning of cellular processes remain challenging. There is a need for a powerful tool, which enables powerful regulation of whole genetic networks and yet is simple to use. In the last years there have been several approaches which were able to alter gene expression, but they have the flaw that they are only targeting one gene at a time and do not allow flexible modification of entire genomes.

In the last 8 months, our team has been planning, developing and building up a universal toolkit, which enables the regulation of complex networks. A toolkit that allows the simple and flexible regulation of multiple genes by only one protein.

Figure 1: Our uniCAS toolkit The uniCAS toolkit provides 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. For multiple targeting different targets can be easily combined using the RNAimer plasmid. Use our custom-tailored Manual Tool to generate your individual manual for your needs of gene regulation.

The great potential harbored by the CRISPR/Cas9 system provided a great basis for us to modify it in a way that allows multiple, sequence specific gene regulation in mammalian cells.
The first step to a universal toolkit was to generate a sequence specific DNA-binding protein. This was archieved by mutating the DNA-cleavage site of the Cas9 herwith termed dCas9 protein. With the catalytically inactive engineered mutant of Cas9 we were able to influence the DNA-binding locus and direct the protein to requested DNA targets. To establish a tool that influences the gene expression in mammalian cells, we fused different effectors to the dCas9 protein. For gene regulation we used the trans-activator VP16, the repressor-domain KRAB and the histone methyltransferase G9a. These effectors were able to efficiently change the expression rate of a reporter protein SEAP (secreted alcaline phosphatase) and the endogenous signal protein VEGF (vascular endothelial growth factor).

Figure 3: Engineering of the CRISPR/Cas9 system The nuclease function of Cas9 has been mutated. The dCas9 is still able to bind the guiding RNAs and therewith can be directed towards every requested sequence. By fusing effectors to the dCas9, gene regulation of specitic loci can be engineered.

In order to allow for control of gene regulation in a time- and space-dependent manner, we designed various control elements. We engineered and tested light induction systems and also tried to activate the expression upon chemical stimuli.

As dCas9 is a relatively large protein consisting of 1367 amino acids and a molecular weight of 160 kDa and thereby influences expression rates and toxicity, we furthermore designed several truncated versions of dCas9 with the aim to minimize size and increase the expression rate with retained binding capacity.

Last but not least we established a novel ELISA-based method to assess the binding efficiency of the Cas9 proteins: The uniCAS Binding Assay “uniBAss”. uniBAss was shown to be an innovative tool for the characterization of the binding capacity of our dCas9 fusion proteins and the truncated dCas9 versions. The method offers the possibility for high throughput capabilities and expansion to DNA-binding proteins others than Cas9 such as TALEs and ZFNs.

Hidden as an uncharacterized E. coli locus for more than 15 years [1] , Barrangou et al. identified the CRISPR (Clusterd Regularly Interspaced Short Palindromic Repeats) array as previously unknown adaptive prokaryotic immune system [2] . Almost half of all prokaryots make use of this defence mechanism. Thereby, invasive DNA can specifically and efficiently be cleaved. It helps controlling the unselective uptake through natural transformation, phage DNA transduction and horizontal gene transfer by conjugation [3] . This unique feature results from the interplay of non-coding RNAs and CRISPR associated proteins (Cas) which are able to cleave invasive DNA. A wide range of different CRISPR subtypes is known [4] , [5] . We used CRISPR type II b of S. pyogenes.

The recognition and degradation of invasive DNA by CRISPR/Cas occurs in three steps (see Fig. 2):

1. Acquisition: Invasive DNA is recognized via a protospacer adjacent motif (PAM) – the sequence NGG. This sequence is then integrated into the host CRISPR array and is named spacer. Spacer sequences transcribe for crRNAs (CRISPR RNAs) which help to cleave sequence specifically invasive DNA. Spacer sequences are located between short palindromic repeats. These palindromic repeats help the functionality of the crRNAs.
2. Expression/Transcription: The Cas9 endonuclease is expressed and spacer sequences are transcribed to crRNAs. crRNAs contain the complementary spacer sequence and the direct repeat sequence. The crRNA is processed by RNAse III and functions as a guide for the Cas9 protein to trace invasive DNA sequence specifically. Furthermore trans-activating crRNAs (tracrRNA) are transcribed and bind to the direct repeat part of the crRNA [6] . This second type of RNA is needed to build a structure that can be bound by the Cas protein.
3. Interference: Appropriate invasive DNA is detected by the RNA-protein-complex. Now the endonuclease Cas9 disposes the invasive DNA by cleavage.

In short: This CRISPR/Cas9 system is based on the interplay of three acting components: tracrRNA, crRNA and Cas9 protein. Studies showed that RNAse III is sufficiently available in host cells [7] . crRNA helps to detect complementary invasive DNA, tracrRNA enables the interaction of crRNA and Cas9. RNA-Cas9 complex binds appropriate DNA and Cas9 cleaves the DNA sequcence specifically.

Unlike Zinkfingers, TAL effectors or Meganucleases, Cas9 proteins perform DNA sequence specific adhesion by utilizing a unique RNA scaffold composed of crRNA and tracrRNA. With about 20 nucleotides corresponding to the target element and the Cas9's helicase activity, Watson-Crick base pairing can be established between crRNAs and the desired DNA. By their short size, crRNAs are easy to order, insert and express from vector plasmids, containing an RNA-Polymerase III driving U6 promoter, like our RNAimer. So far, the only constraint for recognition between crRNA and target DNA is a protospacer adjacent motif - PAM -, located directly 3‘ of the protospacer locus and containing a NGG triplett. A second, trans-acting crRNA - tracrRNA - mediates pre-processing of the crRNA and indispensably enhances formation of the Cas-crRNA ribonucleoprotein complex [6] .

In the near future CRISPR/Cas9 systems can commonly be used to target multiple DNA sequences. Thereby, co-transfecting of standardized crRNA array and tracrRNA encoding plasmids with a Cas9 protein might yield a powerful device for multiplex genome engineering [7] , [8] . It opens up the possibility to deal with a wide range of yet unadressed scientific questions in the near future, including Systems Biology aspects and complex metabolic approaches. Other advantages cover monetary and logistic aspects, as the only component for modification lies indeed within the crRNA itself. In turn, this can be ordered as two corresponding forward and reverse primers - see Team Freiburg's easy crRNA Designing Tool. Financial, logistic and human ressources stay at a minimum. Accordingly, CRISPR/Cas9 systems have already been established for many model organisms, including Saccharomyces cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana, Drosophila melanogaster, Danio rerio and Mus musculus.

References