By Jeyashree Sundaram, MBA
Transcription Activator-Like Effector Nucleases (TALENs) are artificial restriction enzymes that have the ability to cut DNA at the point of contact with a series of nucleotides.
Credit: Sergey Nivens/Shuttershock
TALENs involve the synthesis of the C-terminal with type II restriction enzyme, Fokl, in order to produce heterodimers that form a double-stranded break (DSB) in DNA.
Recent advancements in TALEN include nonspecific DNA-splitting nuclease combined with a DNA binding domain that can be easily designed to facilitate TALENs for effective targeting of various gene sequences.
The capability of TALENs supports the development of biological research and enhances the methods of treatment for genetic diseases.
TALEN techniques based on genome engineering
Genome editing is a new emerging method that allows the direct manipulation of any type of gene belonging to different organisms and cells.
This method is based on the utilization of engineered nucleases that consist of a sequence-specific DNA-binding domain combined with a regular DNA spit module.
These illusory nucleases consist of programmable and sequence-specific DNA-binding modules coupled with a regular DNA cleaved domain that allows accurate and efficient genetic alterations by stimulating the targeted DNA double-strand breaks to induce cellular DNA repair, including error-prone NHEJ and HDR.
Hence, the adaptability and ease of TALENs techniques have led to the development of genetic engineering.
DNA-binding domain
DNA-binding molecules can be merged with several effector domains to influence genomic structures and functions; including nucleases, recombinases, transposases, transcriptional activators and repressors, DNA histone acetyltransferases, and DNA histone methyltransferases. So, the capability of execution of genetic alterations successfully depends on the selective DNA binding and closeness of TALE proteins.
The basic unit used to build the DNA-binding domains of TALENs are highly conserved repetitive sequences acquired from Xanthomonas proteobacteria encrypted TALEs.
A Type III secretion system is utilized to inoculate these TALEs into the cells of the host plants; in order to modify transcription in the host cells, these TALEs are then bound to the genomic DNA. This process permits the colonization of the pathogenic bacteria.
Highly-conserved repetitions of 33-35 amino acid groups that are bound by other TALE-derivatives in the terminal ends of carboxy- and amino-units facilitate the complete DNA-binding mechanism.
Applications of TALENs
The major applications of Transcription Activator-Like Effector Nucleases (TALENs) are discussed below:
Insertion of targeted DNA in potato plants
Addition of targeted DNA into known location of genome is more advantageous than the arbitrary insertion mechanism that involves standard genetic modification methods.
Precise combination of transgenes can assure co-segregation and enhance the prediction of the level of gene expression, which makes the line selection and downstream characterization more useful. The deregulation procedures of transgenic crops are made simpler due to on-site DNA integration.
Various experimental procedures involve the combination of TALEN-induced DSB and non-autonomous marker selection for insertion of transgene into a pre-chosen and transcriptionally active site present in potato genome.
A cytokinin vector is also used in experiments to obtain TALEN expression and to prevent stable integration of nucleases. The results of experiments depict the occurrence of T-DNA stimulated by TALEN, with high frequency and reliable genomic expression.
Genome editing
Genome editing involving TALENs system is widely used in plants. Targeted editing of plant genome can be utilized for investigation of gene function. It can also be used to generate plants with new characteristics such as resistance to pathogens and herbicides, changes in metabolism, productivity etc.
Genome editing in plants using TALENs has been carried out using four model objects.
TALENs has recently evolved as a progressive genome editing tool used in various cells and organisms. The on-site specific chromosomal double-strand breaks induced by TALENs have considerably raised the efficiency of genome modification.
Various uses and values of TALENs
- TALENs are valuable for the formation of knockout strains and for the study of cell mutations of various organisms like yeast, bacteria, rat embryonic stem cells, fish, xenopus, human cell lines, rodents, and plants. Various assays enable the evaluation of the cutting efficiencies of TALENs.
- 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.
- TALENs allow effective induction of targeted alterations in various model organisms that are challenging for genetic manipulation like roundworm, fruit fly, zebrafish, pig, rat and frog. They are also involved in endogenous genomic modification in cells of silkworm, cricket, and cow.
- Recent improvements in TALENs technique support the study of genomes in wheat plants and allow rapid modification in the agronomic properties of the crop. They also help in avoiding the need for non-host DNA. The improvements in crop properties include increased yield and increased immunity against several diseases.
Further Reading