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Transcription Activator-Like Effector Nucleases (TALENs)

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Transcription Activator-Like Effector Nucleases (TALEN) is restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases. Alongside zinc finger nucleases and CRISPR/Cas9, TALEN is a prominent tool in the field of genome editing.

Transcription Activator-Like Effector Nucleases (TALENs) are of a similar construction as ZFNs. They are derived from naturally occurring plant pathogenic bacteria and contain DNA binding proteins called TALEs. The TALE is 33–35 amino acids in length and recognizes a single base pair of DNA. Just like ZFNs, a series of these TALEs are linked together to form a chain capable of targeting a single site within the genome. The nuclease used with TALENs is also commonly FokI. Therefore like ZFNs, a pair of TALENs is used to induce a double stranded break. The ability to recognize a single base, as opposed to a triplet, gives TALENs greater flexibility over ZFNs. Additionally, many effector domains have been made that are able to be fused to TALEN chains for targeted genetic modifications, including transcriptional activators, and site-specific recombinases. However, construction of a TALEN array requires the assembly of multiple, nearly identical repeat sequences, which can be technically challenging. This has led to the development of several novel laboratory methods, specifically to enable the assembly of custom TALEN arrays including Fast Ligation-based Automatable Solid-phase High-throughput (FLASH), Iterative Capped Assembly (ICA) and commercial DNA synthesis.

TALENs and ZFNs have been proposed as alternative platforms for engineering homing-based gene drive systems, that is, systems that spread by cleaving a specific target sequence and then using the cell’s repair machinery to copy themselves to the target site. The benefit of TALENs and ZFNs over HEGs is that they can be easily engineered to target desired DNA sequences due to the modular nature of their DNA-binding domains. The relationship between these repeats and DNA recognition can be exploited to design TALENs that target virtually any desired DNA sequence. For ZFNs, DNA-binding specificity can be similarly manipulated, being determined by an array of finger modules that can be generated either by selection using large combinatorial libraries, or by rational design.

Uses

  • TALEN has also been harnessed to develop tools for the production of biofuels. 
  • It has been used to engineer stably modified human embryonic stem cell and  induced pluripotent stem cell (IPSCs) clones and human erythroid cell lines, to generate knockout C. elegans, knockout rats, knockout mice and knockout zebrafish. Moreover, the method can be used to generate knockin organisms. 
  • TALEN has also been utilized experimentally to correct the genetic errors that underlie disease. Recently, it was shown that TALEN can be used as tools to harness the immune system to fight cancers; TALEN-mediated targeting can generate T cells that are resistant to chemotherapeutic drugs and show antitumor activity. 
  • 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.  
  • 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.  
  • TALENs can be formed to enable induction of mutations into the open reading frames of viruses, such as hepatitis B, HIV, and herpes, which are present in the latent state inside the body and are unaffected by treatment that inhibits viruses and their replications. Hence, their symptoms are permanently evaded with accurate genomic modifications.
  • TALEN has been used to efficiently modify plant genomes, creating economically important food crops with favorable nutritional qualities.

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