Transcription activator-like effector nuclease

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Transcription activator-like effector nucleases (TALEN) are

The non-specific DNA cleavage domain from the end of the FokI endonuclease can be used to construct hybrid nucleases that are active in a yeast assay.^[6][7] These reagents are also active in plant cells^[8][9] and in animal cells.^{[9][10][11][12]} Initial TALEN studies used the wild-type FokI cleavage domain, but some subsequent TALEN studies^[11][13][14] also used FokI cleavage domain variants with mutations designed to improve cleavage specificity^[15][16] and cleavage activity.^[17] The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity.^[10][18]

Engineering TALEN constructs

The simple relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for the efficient engineering of proteins. In this case,

Transfection

Once the TALEN constructs have been assembled, they are inserted into plasmids; the target cells are then transfected with the plasmids, and the gene products are expressed and enter the nucleus to access the genome. Alternatively, TALEN constructs can be delivered to the cells as mRNAs, which removes the possibility of genomic integration of the TALEN-expressing protein. Using an mRNA vector can also dramatically increase the level of homology directed repair (HDR) and the success of introgression during gene editing.

Genome editing

Mechanisms

TALEN can be used to edit genomes by inducing double-strand breaks (DSB), which cells respond to with repair mechanisms.

Non-homologous end joining (NHEJ) directly ligates DNA from either side of a double-strand break where there is very little or no sequence overlap for annealing. This repair mechanism induces errors in the genome via indels (insertion or deletion), or chromosomal rearrangement; any such errors may render the gene products coded at that location non-functional.^[10] Because this activity can vary depending on the species, cell type, target gene, and nuclease used, it should be monitored when designing new systems. A simple heteroduplex cleavage assay can be run which detects any difference between two alleles amplified by PCR. Cleavage products can be visualized on simple agarose gels or slab gel systems.

Alternatively, DNA can be introduced into a genome through NHEJ in the presence of exogenous double-stranded DNA fragments.^[10]

Homology directed repair can also introduce foreign DNA at the DSB as the transfected double-stranded sequences are used as templates for the repair enzymes.^[10]

Applications

TALEN has been used to efficiently modify plant genomes,

In theory, the genome-wide specificity of engineered TALEN fusions allows for correction of errors at individual genetic loci via homology-directed repair from a correct exogenous template.[33] In reality, however, the in situ application of TALEN is currently limited by the lack of an efficient delivery mechanism, unknown immunogenic factors, and uncertainty in the specificity of TALEN binding.^[33]

Another emerging application of TALEN is its ability to combine with other genome engineering tools, such as meganucleases. The DNA binding region of a TAL effector can be combined with the cleavage domain of a meganuclease to create a hybrid architecture combining the ease of engineering and highly specific DNA binding activity of a TAL effector with the low site frequency and specificity of a meganuclease.^[39]

In comparison to other genome editing techniques, TALEN falls in the middle in terms of difficulty and cost. Unlike

The off-target activity of an active nuclease may lead to unwanted double-strand breaks and may consequently yield chromosomal rearrangements and/or cell death. Studies have been carried out to compare the relative nuclease-associated toxicity of available technologies. Based on these studies [18] and the maximal theoretical distance between DNA binding and nuclease activity, TALEN constructs are believed to have the greatest precision of the currently available technologies.^[41]

See also

• Genome editing with engineered nucleases
• Zinc finger nuclease
• Meganuclease
• CRISPR

References