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. Thus we contribute to actual problems of science. We saw a great potential in the CRISPR/Cas9 system and modified it in a way that allows a multiple, sequence specific gene regulation.

The first step on the long journey 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 protein. With the catalytically dead version of Cas9 (dCas9) we are now able to influence the DNA-binding locus and can 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 the reporter protein SEAP and even the endogenous VEGF locus.

Figure 1: Our uniCAS toolkit Our toolkit consists of different effectors and ...

To make sure that signal networks and pathways can be investigated in a time and space dependent manner, we had to implement a control element. Therefore we developed light induction systems and also tried to activate the expression on chemical stimulus.

dCas9 consists of 1367 amino acids, that's a large protein. Because size, expression rate and toxicity correlate, we designed several truncated versions of dCas9 with the aim to minimize size and increase the expression rate but contain the binding capacity.

To be sure that the catalytically dead version, the fusion proteins or the truncations of dCas9 still posses the ability to bind DNA, we established a novel ELISA-based method to assess the binding efficiency of the Cas9 proteins: The uniCAS Binding Assay “uniBAss”. Therewith we could prove that the DNA-binding capacity is maintained. uniBAss is an innovative tool for the biochemical characterization of the binding capacity of our dCas9 fusion proteins and the truncated dCas9 versions with high throughput capabilities.

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 Eubacteria and most Archaea make use of this defence mechanism. Thereby, invasive DNA can specifically and efficiently be cleaved, controlling 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 dispose complementary 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. Furthermore trans-activating crRNAs (tracrRNA) are transcribed and bind to the direct repeat part of the crRNA [6] . This RNA hybrid is processed by RNAse III and functions as a guide for the Cas9 protein to trace invasive DNA.
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.

Unlike Zinkfingers, TAL effectors or Meganucleases, Cas9 proteins perform DNA sequence specific adhesion by utilizing a unique crRNA scaffold. With about 20 nucleotides corresponding to the target element, 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 & 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.

Sources