Transcription activator-like effector nuclease

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Transcription activator-like effector nuclease (talen, TALEN®) is a technology used in gene editing. Transcription activator-like effectors (TAL effectors, TALEs) are now easy to engineer to bind to most desired DNA sequences. The combination of a TAL effector with a DNA cleavage domain creates a talen which cuts the DNA strand via the engineered restriction enzymes at a predictable point. It is possible to introduce talens into living eukaryotes cells to do gene editing or genome editing in situ. This is also termed genome editing with engineered nucleases (GEEN). The first use of GEEN in man produced full remission of refractory leukaemia in a 1 year old child, reported in November 2015.

Contents

Process

TAL effector design

The DNA binding proteins secreted by Xanthomonas bacteria were discovered to have 33-34 amino acids for each binding site to a nucleotide, with only the 12th and 13th amino acids being variable and being able to be specified for nucleotide selectivity. A particular sequence template can now be produced artificially. the ideal sequence template will be of the minimum length to be unique in one particular organisms genome, directed at an invariant portion of that organisms genome and not too long for mutations or other technical issues to prevent usefulness. Obviously the sequence could be directed at a specific mutation in the genome as that might be what is causing a disease.

Talen design

The DNA binding region of a TAL effector can be combined with the cleavage domain of a meganuclease to combine the highly specific DNA binding activity of a TAL effector with the specificity of a meganuclease. Traditional DNA cleavage with nucleases are less specific.

Insertion

Talen constructs are classically inserted into plasmids. The target cells are then transfected with the plasmids. This step remains problematical and the current evolution of the technology is to deliver the talen as mRNA. This removes the possibility of genomic integration of the talen-expressing protein and increases the level of homology directed repair (HDR) and the success of introgression.

Breakage and repair

The induced double-strand breaks (DSB) in DNA will be repaired by normal cell mechanisms. Homologous recombination might occur. But also non-homologous end joining (NHEJ) reconnects DNA but such repairs can be imperfect which results in a dysfunctional protein product. The introduction of DNA fragments at the same time as the talen might allow perfect repairs.