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Team:Freiburg/Project
From 2012.igem.org
Project
Project overview
In 2009, two groups have simultaneously pointed out that each of these repeats specifically binds to one base of the target DNA via two amino acids (aa 12 and 13), named the repeat variable diresidues (RVD) 2. Moreover, it has been shown that DNA binding of these proteins is highly modular, i.e. the number or order of bases in the target DNA can be changed by adjusting the number or order of the repeats in the TAL protein, respectively. It is still unclear, how the sequence of DNA binding modules and TALE activity correlate. The minimal condition for TALE activity is a thymine at the 5’ end of the target sequence. Further target sequence requirements that allow for one TALEN pair binding site every 35 bp (published by the Voytas lab in 20113) have recently been questioned by Reyon et al.4 In summary, it is very likely that you can find a potential TALE binding site in any sequence you want to target. This, obviously, is very promising for biotechnological and clinical applications. Thus, two major classes of TAL Effectors have been created by replacing the natural acidic activation domain either by other transcription factors (TALE-TFs)5 or by – in most cases – a monomer of the non-sequence specific nuclease FokI, resulting in TAL Effector Nucleases (TALENS)6. A pair of TALENs can be designed to bind adjacent DNA sequences in a way that the two monomers are able to form a functional FokI dimer that produces a double strand break (DBS) within the spacer between the TAL-Effectors (see figure 13). Subsequently, the cell repairs the DBS by either non-homologous end joining (NHEJ, which results in indels at the DSB site) or homologous recombination of exogenously added genetic material. TALENs and TALE-TF have successfully been applied for manipulation of a series of genes in different organisms such as yeast7, tobacco3, fruitflies8,worms9, zebrafish10, rats11 and various human cell types, including human stem cells12. TALE technology is a huge revolution in synthetic biology not only because of higher sequence fidelity or less cytotoxicity compared to other DNA binding proteins (first of all zinc fingers). The main advantage is that they can be produced rationally to bind other a DNA sequence of choice, whereas zinc fingers with the desired binding properties need to be selected from a library of fingers. Although open source platforms have been published for zinc fingers13, this technology is therefore generally very costly, time consuming and does not guarantee binding sites for every predefined sequence. So relatively few laboratories could actually afford using the zinc finger technology. Consequently, deciphering the TAL code also resulted in a huge step towards democratizing targeted DNA manipulation14. Moreover, multiple protocols and open source kits have been published by the few most influential labs in the field over the past year, which further popularized TALEs1534. However, we believe that the last step of democratizing precise gene targeting has not been made yet – this hypothesis is corroborated by the fact that the biotech companies Cellectis bioresearch and Invitrogen have launched quite expensive new TAL effector product lines during the last few months. In order to bring TAL technology within reach for everyone, in particular for future iGEM students, we identified the two main bottlenecks of conventional TALE assembly, namely that it is very time consuming and requires substantial training in molecular biology. In the next steps, we invented a new method, called Golden Gate cloning- based, automatable TAL Effector (GATE) assembly, and built the genetic parts (the GATE assembly toolkit) to actually assemble custom TALEs. Furthermore, we quantified the efficiency of our GATE assembly and tested our construct in a Human Embryonic Kidney (HEK) cell line. We are proud to say that with our GATE assembly kit, future iGEM students will be able to easily assemble custom 12.5 repeat TALEs faster than anyone else. Working on the GATE assembly kit, we learned a lot about Golden Gate cloning and came up with a strategy to introduce this powerful cloning technology to the iGEM registry as the Golden Gate standard without compromising [RFC 10] standard. Our major goal was to lay a solid foundation for super-easy site specific genome modifications by future iGEM teams.That is why we dedicated a whole subsection of our project description to a step-by-step GATE assembly protocol. We believe that by enabling virtually anyone to specifically manipulate any locus even in the context of a whole genome, we have done the last step towards democratizing gene targeting. Although to date, the GATE assembly kit is complete for little less than a week, we get requests from research groups in Freiburg almost every day , asking for copies of the kit. We are therefore thinking about giving the kit to the open source plasmid repository Addgene so that it can have a positive impact on the research world around iGEM.
We believe that we have laid a solid foundation for super-easy site specific genome modifications by future iGEM teams.
0. Introduction
1. Golden Gate Standard
2. The TAL Vector
3. GATE Assembly Kit
4. Using the Toolkit
4. The future TAL projects
5. Experiments and Results
