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. That is why this technology is generally very costly, time consuming and does not guarantee binding sites for every predefined sequence, although open source platforms have been published for zinc fingers13. So relatively few laboratories can 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 constructs 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 in the world. 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 empower future iGEM students to use and further develop TALE technology. 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 receive 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

Team:Freiburg/Project/References

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