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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 (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 bases or sequence of 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, fruitflies⁶8,worms⁶9, zebrafish¹⁰, rats¹¹ and various human cell types, including human stem cells¹².

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).
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