"
Team:Freiburg/Project/Intro
From 2012.igem.org
(Difference between revisions)
| (7 intermediate revisions not shown) | |||
| Line 188: | Line 188: | ||
<p><div align="justify">Originally, TAL proteins are virulence factors of the plant-pathogenic ''Xanthomonas spp.'' that are injected into plant cells via a type III secretion system in order to modulate transcription<sup>1</sup>. 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. | <p><div align="justify">Originally, TAL proteins are virulence factors of the plant-pathogenic ''Xanthomonas spp.'' that are injected into plant cells via a type III secretion system in order to modulate transcription<sup>1</sup>. 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. | ||
| - | 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)<sup>2</sup>. Moreover, it has been shown that DNA binding of these proteins is highly modular, i.e. the number of bases or sequence of the target DNA can be altered by adjusting the number or order of the repeats in the TAL protein, respectively. | + | 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)<sup>2</sup> (see figure 1). Moreover, it has been shown that DNA binding of these proteins is highly modular, i.e. the number of bases or sequence of the target DNA can be altered by adjusting the number or order of the repeats in the TAL protein, respectively. |
| - | <img src="https://static.igem.org/mediawiki/2012/ | + | <br><br><img src="https://static.igem.org/mediawiki/2012/0/02/Schemetalprotein.png" width="500px" style="margin-left:130px"/><br><div align="center"><br>Figure 1: Scheme of a TAL protein</div><br><br> |
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 2011<sup>3</sup>) have recently been questioned by Reyon et al.<sup>4</sup> In summary, it is very likely that you can find multiple potential TALE binding sites in any sequence you want to target. | 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 2011<sup>3</sup>) have recently been questioned by Reyon et al.<sup>4</sup> In summary, it is very likely that you can find multiple potential TALE binding sites 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) <sup>5</sup> or by a monomer of the non-sequence specific nuclease FokI, resulting in TAL Effector Nucleases (TALENS).<sup>6</sup> 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 2). <br><br> | 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) <sup>5</sup> or by a monomer of the non-sequence specific nuclease FokI, resulting in TAL Effector Nucleases (TALENS).<sup>6</sup> 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 2). <br><br> | ||
| Line 211: | Line 211: | ||
10. Sander, J. D. et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. ''Nat Biotechnol'' 29, 697–698 (2011).<br> | 10. Sander, J. D. et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. ''Nat Biotechnol'' 29, 697–698 (2011).<br> | ||
11. Tesson, L. et al. Knockout rats generated by embryo microinjection of TALENs. ''Nature Biotechnology'' 29, 695–696 (2011).<br> | 11. Tesson, L. et al. Knockout rats generated by embryo microinjection of TALENs. ''Nature Biotechnology'' 29, 695–696 (2011).<br> | ||
| - | 12. Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. ''Nature Biotechnology'' 29, 731–734 (2011) | + | 12. Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. ''Nature Biotechnology'' 29, 731–734 (2011).<br> |
13. Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7, 171–192 (2012). | 13. Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7, 171–192 (2012). | ||
Latest revision as of 23:02, 26 October 2012
Introduction
+
-
-
October 2009Two research groups publish the TAL Effector codes in the same issue of Science: Amino acid 12 and 13 of every DNA binding module specifically binds to one nucleotide
-
October 2010Voytas Lab develops TALENs. These fusion proteins of FokI and a TAL protein cut as dimers and allow researchers to cut virtually anywhere in the genome. Since double strand breaks increase efficiency of homologous recombination, TALENS are a powerful tool for genetic engineering and gene therapy
-
February 2011Based on an exclusive licensing agreement with the University of Minnasota, Cellectis bioresearch launches its TAL effector product line. One TALEN pair currently costs 5000 Euro (6454 US$, 26.10.12).
-
October 2011The iGEM team from Harvard University employed fancy and expensive techniques to find up to 15 new zinc fingers (each of which binds to 3 bp). There has to be a better way…
-
December 2011Nature chooses TALENs as the 2011 Method of the year.
-
February 2012The first two crystal structures of TALE modules bound to DNA published in the same issue of Science. The protein literally wraps itself around the DNA double helix and forms these beautiful symmetric shapes.
-
April 2012Joung lab publishes FLASH assembly in Nature Biotechnology. This first automatable TAL assembly platform facilitates assembly of 96 TAL DNA fragments in less than a day using a pipeting robot.
-
October 2012The Freiburg iGEM team makes TALE technology available to everyone by introducing the GATE assembly kit. For TALEs targeting 14 bp, this platform is currently the fastest, cheapest and easiest method in the world.
Originally, TAL proteins are virulence factors of the plant-pathogenic ''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.
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 (see figure 1). Moreover, it has been shown that DNA binding of these proteins is highly modular, i.e. the number of bases or sequence of the target DNA can be altered by adjusting the number or order of the repeats in the TAL protein, respectively.
Figure 1: Scheme of a TAL protein
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 multiple potential TALE binding sites 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 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 2).
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. That way, TALENs allow researchers to introduce genes into a genome with much higher efficiency than before. In this context, TALENs and TALE-TF have successfully been applied for manipulation of a series of genes in different organisms such as yeast7, tobacco3, fruitflies6,8,worms6,9, zebrafish10, rats11 and various human cell types, including human stem cells12. Moreover, TALENs are already applied for gene therapy in preclinical trials.
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. Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7, 171–192 (2012).
Back to top
Figure 1: Scheme of a TAL protein
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 multiple potential TALE binding sites 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 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 2).
Figure 2: Schematic drawings of a TAL-TF and a TALEN pair13
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. That way, TALENs allow researchers to introduce genes into a genome with much higher efficiency than before. In this context, TALENs and TALE-TF have successfully been applied for manipulation of a series of genes in different organisms such as yeast7, tobacco3, fruitflies6,8,worms6,9, zebrafish10, rats11 and various human cell types, including human stem cells12. Moreover, TALENs are already applied for gene therapy in preclinical trials.
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. Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7, 171–192 (2012).
Back to top
