Team:Freiburg/Project

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Project


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Overview

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 (e.g. 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 fingers1. 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 manipulation2. 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 TALEs3,4,5. 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 [http://www.addgene.org/ 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 for future iGEM teams.

Introduction

You don't know what TAL effectors actually are? We reviewed the recent literature for you, to give you a quick overview of this exciting new field of research.


Golden Gate Standard

Assembling multiple gene constructs in frame without leaving scars is not possible with existing iGEM standards. We therefore introduce the new Golden-Gate Standard that is fully compatible with RFC 10.


The TAL Vector

Targeting a sequence and not doing something to it, is like throwing mechanics at your car. Your car will not get any better only the mechanics will get mad. Because we know this, we bring the tools you need to actually work with DNA.To make it even more easy these tools are deliverd already inside the final TAL backbone, just add the sequence and you're ready.


GATE Assembly Kit

We have invented a super-fast, super-easy and super-cheap Method for custom TAL effector construction. Learn about the theory behind the TAL effector toolkit, how we created it and why we choose this design.


Using the Toolkit

Our overall goal is to empower future iGEM teams to use the most exciting new technology synthetic biology has to offer. We therefore not only invented the GATE assembly platform but wrote a step by step manual for super-easy custom TALE construction


The Future of TAL

Until now, almost three years after deciphering the TALE code, only two types of TAL Effectors have been developed: TALENs and TAL-TFs. We herein propose additional classes of TAL effectors.


Experiments and Results

We not only rigorously tested if our in vitro TALE gene assembly method works but also if our TALE constructs actually work in a human cell line. Check out test design 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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