Introduction

TALENs have recently swooped down and snatched the genome editing monopoly from ZFNs. TALENs works almost the same way as ZFNs. TALENs is a fusion of transcription activator-like (TAL) proteins with FokI nucleases. The TAL protein consists of 33-34 amino acid repeat patterns, with two variable positions, and has a strong ability to identify specific nucleotides. Specific cutting of the genome can be achieved by assembling these TLL arrays and incorporating them into FokI nucleases. When two TALENs are combined and meet, the FokI domain induces a double-stranded break, which can cause the gene to become inactive or can be used to insert the DNA of interest. Tallun is more specific than ZFN, and the monomer has no cross-reaction problems that plague ZFN researchers. This simplicity facilitates the automation of THE TALEN structure. On the downside, because each nucleotide requires a TAL, TALEN is larger and more difficult to deliver than ZFN.

Transcription activator-like nuclease technology utilizes artificially restricted enzymes produced by integrating the TAL effector DNA binding domain into the DNA cleavage domain.

Restrictive enzymes are enzymes that cut DNA strands under a specific sequence. Transcription activator sample effectors (TALEs) can be designed quickly to bind almost any desired DNA sequence. By combining this engineering TALE with the DNA cleavage domain (cutting DNA strands), one can design limit ingasers that specifically cut any desired DNA sequence. When these restrictive enzymes are introduced into cells, they can be used for gene editing or in situ genome editing, a technique known as genome editing with engineered nucleases. In addition to zinc finger nucleases and Cas9 proteins, TALEN is becoming an important tool in genomic editing.

TAL effector DNA-binding domain
TAL effectors are proteins that are secreted by Xanthomonas bacteria. The DNA binding domain contains a repeating highly conservative sequence of 33-34 amino acids, with the 12th and 13th amino acids of divergence. These two locations, called the repeated variable binary (RVD), are highly variability and have a strong correlation with specific nucleotide identification. This relationship between amino acid sequences and DNA recognition allows specific DNA binding domains to be engineered by selecting combinations of repeating segments that contain the appropriate RVD.

DNA cleavage domain
Nonspecific DNA lysis domains starting at the end of FokI in the divide enzymes can be used to build hybrid nucleases that are active in many different cell types. The FokI domain acts as a diplet and requires two constructs with unique DNA binding domains for sites with the right direction and spacing in the target genome. The amount of amino acid residue between the TALE DNA binding domain and the FokI lysate domain, as well as the alkali base between the two separate TALEN binding sites, appear to be important parameters for achieving high levels of activity.

Figure 1. Use TALEN technology to edit the genome workflow of your favorite genes (YFG). The target sequence is identified, a corresponding TALEN sequence is engineered and inserted into a plasmid. The plasmid enters the target cell, where it is converted to produce a functional TALEN, which enters the nucleus and binds and fits into the target sequence. Depending on the application, this can be used to introduce errors (knockout the target gene) or introduce new DNA sequences into the target gene.

TALEN mechanism
The simple relationship between amino acid sequences and THE DNA recognition of the TALE binding domain allows for effective engineering of proteins. Once the TALEN structure is assembled, they enter the plasmids, then transfect the target cells with plasmids and express the gene products and enter the nucleus into the genome. Alternatively, the TALEN structure can be delivered to cells in the form of mRNA, eliminating the possibility of genome integration of TALEN expression proteins. The use of mRNA vectors can also significantly improve the level of homologous directional repair (HDR) and successfully introvert during gene editing.

TALEN technology can be used to edit the genome by inducing double-stranded fracture (DSB), which cells respond to by repairing the mechanism. Non-homogenous end connections (NHEJs) reconnect DNA from either side of a double-stranded break, where the annealed sequence overlaps little or no. This repair mechanism causes errors in the genome by insertion or deletion or chromosomal rearrangement; Because this activity may vary depending on the species used, cell type, target gene, and nuclease, it should be monitored when designing a new system. Alternatively, DNA can be introduced into the genome through NHEJ in the presence of exogenous double-stranded DNA fragments. Homologous directional repair can also introduce foreign DNA in DSB, as transfected double-stranded sequences are used as templates for repair enzymes.

Gene Therapy Application
TALEN technology has been used to efficiently design the steady-state transformation of human embryonic stem cells and induced pluripotent stem cell (IPSC) cloning and human red blood cell lines. The technique has also been used in experiments to correct underlying genetic errors in the disease. For example, it has been used in vitro to correct genetic defects that cause disease, such as knife cell disease, xeroderma pigmentation, and epidermal hemolytic bovine sands. It has also been shown that TALEN technology can be used as a tool to use the immune system to fight cancer. Theoretically, engineering TALEN fusion genome-wide specificity allows for the correction of individual gene sites by directing repair from the correct exogenous template. However, in reality, the field application of TALEN ® technology is limited by the lack of an effective transmission mechanism, unknown immunogenicity factors and the uncertainty of TALEN binding characteristics. Another emerging application of TALEN ® technology is its ability to bind to other genomic engineering tools, such as gene enzymes. The DNA binding region of the TAL effector can be combined with Megan Neckele's cleavage domain to create a hybrid architecture that combines the engineering simplicity of the TAL effector with a highly specific DNA binding activity with a low point frequency and specificity of a Megancil.

In 2015, doctors at Great Ormond Street Hospital announced the first clinical use of TALEN-based genome editing. An 11-month-old infant with CD19-plus acute lymphoblastic leukemia was treated with modified donor T cells designed to attack leukemia cells, become resistant to Alemtuzumab, and evade detection by the host immune system after introduction. After a few weeks of treatment, the patient's condition improved; though physicians are cautious, the patient has been in remission for several months following treatment.

TALEN Additional Reading

Sun, N., and Zhao, H. (2013). Transcription activator-like effector nucleases (TALENs):

a highly efficient and versatile tool for genome editing. Biotechnol. Bioeng. 110, 1811-1821.

This review examines the mechanism of TALEN system function. The authors also provide a list of papers that have used TALENs in various organisms, describe the biochemical function of the system, and discuss challenges and future perspectives.

Wei, C., Liu, J., Yu, Z., Zhang, B., Gao, G., and Jiao, R. (2013). TALEN or Cas9 – rapid, efficient and specific choices for genome modifications. J. Genet. Genomics 40, 281-289.

This review compares the TALEN and CRIPSR systems. The functional differences and relative strengths a weakness of each method are discussed.

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