Gene editing tools-artTALEs

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Gene editing tools-artTALEs

Post by Acilegna »

Artificial Transcription Activator - like Effector (artTALEs)

What about TALEs?
TALEs are proteins synthesized and secreted by Xanthomonas axonopodis pv. manihotis (Xam), that bind to the promoter region of the genes of the infected plant cell and interact with the transcriptional machinery. That is, they act as a transcription factor. The way that TALEs can interact with a plant's DNA is due to the particular organization that allows them to recognize the promoter sequence of the plant's genes.

Structure of TALEs
The main components of TALE are (1) nuclear localization signals (NLS) that allow going to the nucleus of the plant cell, (2) an acidic domain (AD) of transcription activation that allows it to behave as a transcription factor and (3) a DNA binding domain that has 33-35 tandem repeating amino acids. Amino acids at positions 12 and 13 are highly variable and are called repeat variable di-residues (RVDs). Thus, the number of repeats of the 34 amino acids and the RVDs determine the target nucleotide sequence in the promoter region to which the TALE binds. The region to which RVDs bind is called EBE (Effector Binding Elements).
TALE structure.jpg
TALE structure.jpg (22.46 KiB) Viewed 185 times
Structure of TALE. Boch et al. (2009).

Due to the modular architecture, the mechanism by which TALEs bind to the promoter sequence of host cell genes and the RVD-base specificity code (NN = G or A, HD = C, NH = G, NI = A, NG = T), it has been possible to design and construct artificial TALEs (artTALEs) and use them as a tool to induce target gene expression

The mechanism by which TALEs bind to the promoter sequence of host cell genes has been used as a tool to induce target gene expression by constructing artificial TALEs (artTALEs) or nucleases TALEs (TALENs). When they are introduced into an organism they can modify the genetic expression of an organism's cells by inducing the transcription of target genes or make specific cuts from the use of restriction enzymes (nucleases). The use of these proteins in gene editing could also be exploited in organisms such as humans and in the treatment of genetic diseases such as cancer.

Design of artTALEs
First of all, we must obtain the sequence of the promoter region (1 kb upstream of start codons) of the gene of interest using databases like NCBI or Phytozome. Then, we will introduce the promoter sequence to one of the tools available online (TALE-NT, TALVEZ, Storyteller, TALgetter, Target Finder, or PrediTALE) to predict the amino acid sequence of potential artTALEs and their EBEs. If we use the TALVEZ tool, the artTALEs that are closest to the transcription start site (TSS) will be chosen, with a score > 12 and that the difference between the score of the first EBE and the second EBE of the same artTALE is ≥ 4 to guarantee the specificity of the artTALE with the chosen sequence.

Assembly of artTALE modules
It depends on how big we want to make our artTALE (more repeat domains), the amino acid sequence of the artTALE will be divided into sub-modules to carry out the assembly in assembly vectors and finally in a single expression vector. For example, if we want to build an artTALE of 18 repeats, that is, it will bind to a sequence of 18 base pairs in DNA, we will divide the artTALE into three modules of six repeats.
Golden TAL Technology is one of the cloning strategies that work as a toolbox for assembling artificial TALEs that bind to DNA sequences in the promoter region of a specific gene. Golden Tal Technology is based on Golden Gate cloning to build a library of repeats that in turn will be used in the construction of each module, like Lego blocks. Finally, each module will be assembled into a final expression vector in order to obtain the complete artTALE.
It is necessary to take into account that in addition to the repeats that will bind to the DNA, it is necessary to add the vectors with the corresponding N-terminal and C-terminal. Moreover, it will be required to add a tag to confirm that the artTALE is located in the nuclei of the cells of the organism to which it will be introduced, for example, in a plant. This tag is usually a green fluorescent protein (GFP).
journal.pone.0019509.s005.jpg (755.84 KiB) Viewed 166 times
Golden TAL Technology toolbox. Geißler et al. (2011).

Boch et al. (2009). doi: 10.1126/science.1178811
Doyle et al. (2012). doi: 10.1093/nar/gks608
Geißler et al. (2011). doi:10.1371/journal.pone.0019509
Niño A, López C (2020)
Pérez-Quintero et al. (2013). doi:10.1371/journal.pone.0068464
Sanjana et al. (2012). doi:10.1038/nprot.2011.431
Streubel et al. (2012). doi:10.1038/nbt.2304
Streubel et al. (2013). doi:10.1111/nph.12411
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