Team:Freiburg/Project

From 2012.igem.org

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<div class="issuedate">Discovery of the zinc finger protein</div>
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<p>Jonathon Miller, A. D. McLachlan, and Sir Aaron Klug first identify the repeated binding motif in Transcription Factor IIIA and are the first to use the term ‘zinc finger.'</p>
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<p>Jonathon Miller, A. D. McLachlan, and Sir Aaron Klug first identify the repeated binding motif in Transcription Factor IIIA and are the first to use the term ‘zinc finger.'</p>
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<div class="issuedate">First crystal structure of a zinc finger</div>
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<p>Carl Pabo and Nikola Pavletich of Johns Hopkins University solve the crystal structure of zif268, now the most-commonly studied zinc finger. This paved the way for construction of binding models to describe how zinc fingers bind to DNA, setting the foundation for future custom engineering of zinc finger proteins.</p>
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<div class="issuedate">CEO Edward Lanphier founds Sangamo Biosciences</div>
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<p>Edward Lanphier leaves Somatix Therapy Corporation and makes a deal for exclusive rights to the work of Srinivan Chandrasegaran of Johns Hopkins University who combined the Fok I nuclease with zinc fingers.</p>
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<div class="issuedate">Srinivasan Chandrasegaran publishes work on fusing the Fok I nuclease to zinc fingers</div>
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<p>By attaching nuclease proteins to zinc fingers, a new genome editing tool was created. The DNA-binding specificity of zinc fingers combined with the DNA-cutting ability of nucleases opened up possibilities for future research in gene therapy by allowing researchers to directly modify the genome though use of zinc finger nucleases.</p>
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<div class="issuedate">Sangamo enters the public sector</div>
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<p>In April 2000, five years after its founding, Sangamo Biosciences goes public offering 3.5 million shares at a starting value of $15 per share.</p>
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<div class="issuedate">Sangamo patents zinc finger nuclease technology</div>
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<p>Sangamo's patent, titled "Nucleic acid binding proteins (zinc finger proteins design rules)", ensures that any use or production of zinc fingers with attached nucleases is the intellectual property of Sangamo.</p>
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<div class="issuedate">Rapid open source production of zinc finger nucleases becomes available</div>
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<p>Researcher Keith Joung of Harvard University and Mass. General Hospital develops a method for making zinc finger nuclease proteins that bind to custom target sequences, utilizing a bacterial two-hybrid screening system to identify specific zinc finger binders to a DNA sequence of interest.</p>
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<div class="issuedate">Zinc finger nuclease enters clinical trials</div>
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<p>Sangamo and University of Pennsylvania begin clinical trials with a zinc finger nuclease designed to target the CCR5 gene and inhibit HIV. Success of this therapeutic could prove a significant advance for gene therapy. </p>
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<div class="issuedate">Context-dependency improves open-source zinc finger engineering</div>
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<p>Keith Joung publishes tables of zinc finger binding sites that account for context-dependent effects and can be rearranged to form custom zinc finger proteins that bind to a variety of DNA sequences. This greatly increases the ease of engineering novel zinc fingers based on the structures of previously characterized zinc fingers.</p>
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<div class="issuedate">Harvard iGEM develops a novel method to engineer custom zinc fingers</div>
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<p>Using novel integration of existing technologies, we have developed a rapid, comparatively low-cost, <strong>open source</strong> method for making thousands of custom zinc fingers by integrating MAGE, lambda red, and chip-based synthesis technologies. Our work greatly increases the ease of access to zinc finger technology for researchers worldwide.</p>
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Revision as of 18:07, 26 September 2012






Project



Projectsymbol.png


Project overview

Originally, TAL proteins are virulence factors of the plant-pathogen Xanthomonas spp. that are injected into plant cells via a type III secretion system in order to modulate transcription1. For this purpose, their c-terminal end contains a nuclear localisation signal (NLS) and an acidic activation domain. The central part of the TAL protein contains a number of almost similar repeats that mediate specific binding to target loci in the genome (see figure 10).

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

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.


1. 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].


2. 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.


3. 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.


4. 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


4. The future TAL projects

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.


5. 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.

Team:Freiburg/Project/References

1. Scholze, H. & Boch, J. TAL effectors are remote controls for gene activation. Current Opinion in Microbiology 14, 47–53 (2011).
2. Moscou, M. J. & Bogdanove, A. J. A Simple Cipher Governs DNA Recognition by TAL Effectors. Science 326, 1501–1501 (2009).
3. 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).
4. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nature Biotechnology 30, 460–465 (2012).
5. Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nature biotechnology 29, 149–153 (2011).
6. Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. Nature Biotechnology 29, 143–148 (2010).
7. Boch, J. et al. Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors. Science 326, 1509–1512 (2009).
8. Liu, J. et al. Efficient and Specific Modifications of the Drosophila Genome by Means of an Easy TALEN Strategy. Journal of Genetics and Genomics 39, 209–215 (2012).
9. Wood, A. J. et al. Targeted Genome Editing Across Species Using ZFNs and TALENs. Science 333, 307–307 (2011).
10. Sander, J. D. et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotechnol 29, 697–698 (2011).
11. Tesson, L. et al. Knockout rats generated by embryo microinjection of TALENs. Nature Biotechnology 29, 695–696 (2011).
12. Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nature Biotechnology 29, 731–734 (2011).
13. 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).
14. Clark, K. J., Voytas, D. F. & Ekker, S. C. A TALE of two nucleases: gene targeting for the masses? Zebrafish 8, 147–149 (2011).
15. Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7, 171–192 (2012).



Zinc Finger Historical Timeline

  • Discovery of the zinc finger protein

    Jonathon Miller, A. D. McLachlan, and Sir Aaron Klug first identify the repeated binding motif in Transcription Factor IIIA and are the first to use the term ‘zinc finger.'