Application of our uniCAS toolkit


Our toolkit opens up the possibility to deal with a wide range of yet unadressed scientific questions in the near future, including the field of systems biology and complex metabolic engineering 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 Design Tool. Logistic, financial and human ressources stay at a minimum using the uniCas toolkit for gene regulation. 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.

Our toolkit inspired leading scientists from the University of Freiburg, providing a cornucopia of possibilities! In the following, we want to further point out some possible application techniques.

Application Ideas

Stem Cell Reprogramming

Notably in 2012, the Nobelprize for Medicine was awarded to Shinya Yamanaka and John Burdon, two outstanding experts and pionieers in stem cell research. In a 2006 and 2007 Cell paper, Yamanaka and his colleagues described an approach to reprogram somatic fibroblast cells of both mice and humans to what they termed "induced Pluripotent Stem Cells" (iPSC) [1][2]. In their precise methodology, the Japanese research group was able to define four essential transcription factors in early embryonic development - Oct4, Sox2, Kif4 and c-Myc. Under exogenously and combinatorial retroviral integration, these were shown to enable dedifferentiation of mature cells in certain tissues.

However, the techniques set up by Yamanaka et al. have not passed certain constraints: Firstly, dedifferentiation rates of their cells did not exceed numbers beyond 0.1% of the original fibroblast population. Furthermore, epigenomic and transcriptomic comparisons between iPSC clones and truly Embryonic Stem Cells (ESC) revealed that the dedifferentiation profile of the obtained pluripotent cells have been at least questionable. The fact that combinatorial transcription factor introduction into somatic cells strictly relied on viral integration also gave significance to serious secondary disadvantages of the method - by hardly controllable target randomness of the utilized viral integrase, the risk of carcinogenic mutations or disruptions of vital host housekeeping genes has lastly become apparent.

However for the last six years since 2007, techniques expanded rapidly. In a stunning research article from 2011, Anokye-Danso et al. from the University of Pennsylvania described a first successful attempt to reprogram human fibroblast cells without any introduction of transcription factors [3]. Instead, their approach consisted of solely retrovirally transducing a combined microRNA-cluster. Thereby, efficiencies of iPSC generation have tremendously raised by two orders of magnitude (100-fold). A second and more demanding task, to dedifferentiate somatic mature cells without causing integrational mutagenesis and by the help of episomal plasmids, is being dealt with in many laboratories - including Yamanaka's [4].

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

What does it have to do with uniCAS? Efficiently up- or downregulating several genomic loci at once is exactly the task that the tool aims at. What about simultaneously upregulating the OKSM promoters or microRNA clusters on a transient basis? Different stoichometric proportions of the involved factors need to be assessed - by targetting more or less protospacers upstream of a gene of interest, this is well possible - so let's take the uniCAS RNAimer for future attempts!

In summary, with novel and efficient methods to create human iPSC, the futuristic vision of personalized medicine and in-vivo tissue regeneration has entered the next stage. Only to mention a few perspectives: from organ transplantations without the risk of severe immune rejections, via pancreatic cell recovery for diabetics, to therapies of certain myeloma types - great hope is being raised by the above mentioned technologies.


(1) Takahashi, K. et al. (2006). Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by defined Factors. Cell 126, 663-667.
(2) Takahashi, K. et al. (2007). Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by defined Factors. Cell 131, 861-72.
(3) Anokye-D., F. et al. (2011). Highly efficient miRNA-mediated Reprogramming of Mouse and Human Somatic Cells to Pluripotency. Cell Stem Cell 8, 367-388.
(4) Okita, K. et al. (2011). A more efficient Method to generate integration-free human iPS Cells. Nature Methods 8, 409-414.

Tissue engineering

The idea of engineered tissues emerged in the mid 1980s to solve the problem of donor-shortage. Nowadays, the field of tissue engineering unifies the work of many life-sciences disciplines, e.g. developemental biology, synthetic biology, systems biology, nanotechnology in terms of scaffold production and many more. Engineered tissues can take many forms, from simple aggregations up to whole organs there is a cornucopia of possibilities. Newly grown tissues can be constructed from donor cells given by the patient leading to great compatibility making the lifelong uptake of immunsuppressiva unnecessary. But these developments are still in their infancy, although first clinical tests generated promising results [5].

… in the near future, human tissue - such as skin or heart - could be renewed by stem cell reprogramming. Theoretically, signaling pathways can be elegantly routed by introducing RNAs - one of them coding for a dCas9-effector - into cells of a patient..." - Prof. Dr. Wolfgang Driever

New developments in tissue engineering are closely linked to stem cells, stem cell differentiation and cultivation of stem cells. Yan Chen et al. and others (see "stem cells") have shown that only few genes are responsible for proliferation and differentiation of cells in vivo, in situ and in vitro. A big challenge is for example the establishment of a vascular system of blood vessels to supply tissues with nutritions and oxygen. Virtually any tissue larger than 0,1 mm needs such a vascular system [6]. One of the factors that leads to the creation of such blood vessels is the Vascular Endothelial Growth Factor (VEGF). It has been shown that its expression is controllable using the Toolkit of iGEM - Team Freiburg 2013.

Another problem is the lack of knowledge about the composition of growth factors, extracellular molecules and scaffold driving the proliferation and differentiation of different tissues. The possibility to finetune gene networks may be crucial in prospective research.

Our toolkit enables researchers around the world to get a better idea of complex intra- and intercellular networks. The uniCas toolkit is a first step towards the goal of understanding and ultimatively controlling cell fate.


(5) Dolgin, E. (2011). Taking tissue engineering to heart. Nature Medicine 17, 1032-1035.
(6) Khademhosseini, A. et al. (2009). Progress in Tissue Engineering. Scientific American, 65-71.