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Transcription Activator-Like Effectors Nucleases (TALENS) - An Outline


Transcription Activator-Like Effectors (TALEs) are proteins secreted by Xanthomonas, a gram-negative bacteria and economically important plant pathogen. During infection, TALEs bind promoter sequences in the host genome in order to activate the expression of  genes that aid infection.

TALEs contain a domain consisting of a repeated sequence that is usually 34 residues in length (Boch and Bonas, 2010). This sequences is conserved except for the the residues at the 12th and 13th positions, which are  known as the repeat variable diresidue (RVD).  The identity of these two residues is directly related to the  target sequence of the DNA bases in host (Boch et al., 2009 ; Moscou et al., 2009) and can therefore be recoded to recognise user-defined sequences.

  • NI = A
  • HD = C
  • NG = T
  • NN = R (G or A)
  • NS = N (A, C, G, or T)
  • NK can also target G although is less active  NN (Miller et al., 2010; Morbitzer et al., 2010)

The second residue of the RVD makes a sequence-specific contact withe DNA, while the first residue stabilises the RVD-containing loop. Target sites of TAL effectors usually contain a T  5’  of the base targeted by the first repeat. This T makes contact with a conserved tryptophan in the region N-terminal of the central repeat domain.

Like zinc-finger nucleases (ZFNs), Transcription Activator-Like Effectors Nucleases (TALENS) are fused to a nuclease domain. The most commonly used nuclease domain is the DNA cleavage domain from the restriction endonuclease FokI. Since FokI is active as a dimer, a pair of TALENS are usually delivered, each designed to recognise the sequences that flank the desired cut site.

TALENS are most easily assembled using Golden Gate cloning as described by Cermak et al (2011). The plasmids required to build TALENS can be obtained from AddGene. The TALENT 2.0 site (Cornell University) hosts software and guides for designing TALEs.

 

References

Boch J, Bonas U (2010). “XanthomonasAvrBs3 Family-Type III Effectors: Discovery and Function”. Annual Review of Phytopathology 48: 419–36

Boch J, Scholze H, Schornack S et al. (2009). “Breaking the code of DNA binding specificity of TAL-type III effectors”. Science 326(5959): 1509–12

Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF (2011) “Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.” Nucleic Acids Res.

Moscou MJ, Bogdanove AJ (December 2009). “A simple cipher governs DNA recognition by TAL effectors”. Science 326 (5959): 1501

Miller, J. C.; Tan, S.; Qiao, G.; Barlow, K. A.; Wang, J.; Xia, D. F.; Meng, X.; Paschon, D. E.; Leung, E.; Hinkley, S. J.; Dulay, G. P.; Hua, K. L.; Ankoudinova, I.; Cost, G. J.; Urnov, F. D.; Zhang, H. S.; Holmes, M. C.; Zhang, L.; Gregory, P. D.; Rebar, E. J. (2010). “A TALE nuclease architecture for efficient genome editing”. Nature Biotechnology 29 (2): 143–148.

Morbitzer, R.; Romer, P.; Boch, J.; Lahaye, T. (2010). “Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors”. Proceedings of the National Academy of Sciences 107 (50): 21617–21622