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TALE technology currently revolutionizes synthetic biology, not only because of higher sequence fidelity or less cytotoxicity compared to other DNA binding proteins (e.g. zinc fingers). The main advantage is that they can be produced rationally to bind 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 TAL technology is generally much less costly, time consuming and does guarantee binding sites for every predefined sequence (although open source platforms have also been published for zinc fingers¹). Consequently, deciphering the TAL code also resulted in an enormous step towards democratizing targeted DNA manipulation². Moreover, multiple protocols and open source kits have been published by the most influential labs in the field over the past year, which further popularized TALEs^3,4,5. However, we believe that the last step of democratizing precise gene targeting has not been made yet – this 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 method, that we refer to as Golden Gate cloning- based, automatable TAL Effector (GATE) assembly, and built the genetic parts (the GATE assembly toolkit) to actually assemble custom TALEs at record speed. 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. While 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 existing standards. 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 (including a video tutorial). We believe that by enabling virtually anyone to specifically manipulate any locus even in the context of a complex genome, we have done the last step towards democratizing gene targeting. Although to date, the GATE assembly kit is complete for only a few weeks, we regularly receive requests from research groups all over Europe, asking for copies of the kit. Moreover, we got approached by the open source plasmid repository Addgene that wants to distribute our toolkit. We are currently preparing to send our kit to them so the GATE kit will be available to everyone soon! That way, we have a significant impact also on the research world around iGEM.

We believe that we have laid a solid foundation for super-easy site specific genome modifications for future iGEM teams.

Introduction

Golden Gate Standard

The TAL Vector

GATE Assembly Kit

Using the Toolkit

The Future of TAL

Experiments and Results

References

1. Maeder, M. L. et al. Rapid ‘Open-Source’ Engineering of Customized Zinc-Finger Nucleases for Highly Efficient Gene Modification. Molecular Cell 31, 294–301 (2008).
2. Clark, K. J., Voytas, D. F. & Ekker, S. C. A TALE of two nucleases: gene targeting for the masses? Zebrafish 8, 147–149 (2011).
3. Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7, 171–192 (2012).
4. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39, e82 (2011).
5. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nature Biotechnology 30, 460–465 (2012).

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