Publish Your Research Online
Get Recognition - International Audience
Request for an Author Account | Login | Submit Article
|HOME||FAQ||TOP AUTHORS||FORUMS||PUBLISH ARTICLE|
TALENs: A Reliable Genome Editing ToolBY: Rhitu Rai | Category: Biotech-Research | Submitted: 2017-02-07 20:01:44
Article Summary: "The transformation from randome mutagenesis approach to efficient and specific gene targeting has been enabled by use of engineered nucleases. The article talks about its mechanism, principle and application. Further, TALENs classifies high for reliability due to its site specificity and low off target effects..."
TALEN- A reliable genome editing tool
Authors: Chandrika Ghoshal, Rohini Sreevathsa, Rhitu Rai
National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi-110012
Genome editing is a molecular biology approach which enables researchers to make desired alterations in the genome sequence by precise site specific addition, replacement, or removal of DNA bases in a more efficient manner than conventional genetic engineering. Cell's inherent ability to counterfeit any mutation by utilising their own repair mechanisms has been harnessed to develop the tools for genome editing.
Genome editing involves generation of a Double Strand Break (DSB) in a specific genomic target sequence followed by incorporation of desired modification during subsequent DNA repair. Cells repair a DSB either by homologous recombination (HR), or, non homologous end joining (NHEJ). HR based genome editing utilizes a donor DNA bearing similarity to the sequence as break site as a template. If the donor DNA and break site are in the same cell, homologous recombination between the two homologous sequences come into play amounting to their exchange. This allows direct change of genetic information in cells by introducing a correct version of a DNA sequence to replace an unwanted gene resulting in gene replacement. In the absence of any homologous template, cell can employ various enzymes to directly join the two ends of the DNA break back together. This process is known as non homologous end-joining. Since, this gluing back together is often imprecise, it leads to incorporation or deletion of few bases at the break site. This way a gene with undesired trait can be defunct.
The two major components this technology relies on are: 1. DNA binding domain- customizable sequence specific proteins for identifying the target in genome and binding; 2. Nuclease: for creating a DSB at the target site identified by DNA binding domain in non-sequence specific manner. These two are fused together to work as engineered nucleases for genome editing.
The first genome editing tool, Zinc finger, was discovered back in 1991 which was predominant for over ten years. Then in 2009, TALEN technology emerged as an alternative to ZFNs where the assembly of customizable zinc fingers was not easy.
Transcription Activator Like Effector Nucleases(TALENs) originated from the plant pathogenic bacteria Xanthomonas interaction with plants. Xanthomonads inject type III system secreted proteins called TALE inside the nucleus to modulate the host plant gene expression, thus acting as transcription factors. TALEs have a typical structure with conserved N and C terminal. The specificity is derived from the central region composed of variable number of conserved repeats. Each repeat differs from another at the 12th and 13th residue known as repeat-variable di-residues (RVDs). These TALE repeats help to recognize specific DNA sequences. The sequence of these RVDs in a TALE makes a code to recognise its DNA target in host genome. Each RVD binds to one nucleotide in DNA target. . The first amino acid residue in the RVD (H and N) of TALE is responsible for stabilizing the spatial conformation. The second amino acid residue interacts with one nucleotide. Most studies use monomers containing RVDs such as Asn and Ile (NI), Asn and Gly (NG), two Asn (NN), and His and Asp (HD) for binding the nucleotides A, T, G and C respectively. The amino acids D and N form hydrogen bonds with nitrogenous bases, and I and G bind target nucleotides through van der Waals forces. The HD repeat binds to C, Ni to A, NK to G, NG to T while NS is degenerate and can bind to A, C, G or T. Therefore, the RVDs are customizable and can be formed following the code. TALEs fused to Fok1 endonuclease are called TALENs. Since, the FokI nuclease needs to dimerize for function, the TALE domain is designed in pairs to span the target site. Their binding sites are chosen so that they are located on opposite DNA strands and are separated by small fragments (12-25 bp) of spacer sequence. Once in the nucleus, artificial nucleases bind to target sites and the C terminal FokI domains dimerize to cause a double-strand break in a spacer sequence. TALE repeats can be combined to recognize virtually any user-defined sequence.
TALEN provides greater design flexibility than ZFNs further facilitated by strategies like 'Golden Gate' molecular cloning, high-throughput solid-phase assembly, and ligation-independent cloning techniques. The only targeting limitation for TALE arrays is that TALE binding sites should start with a T base. However, this limitation has also been overcome by selection of mutant variants of the TALEN N-terminal domain that are capable of binding to A, G, or C.
TALENs have been used in a variety of organisms for gene targeting. TALENs are capable of correcting the underlying cause of the disease like sickle cell anemia, xeroderma pigmentosum, epidermolysis bullosa, and permanently eliminate the symptoms with precise genome modifications. TALEN-mediated targeting can generate T cells that are resistant to chemotherapeutic drugs and show anti-tumour activity. It is also used to develop tools for bio fuel production. TALENs can also introduce targeted modifications in several model organisms like C. elegans and zebrafish. TALENs have also allowed researchers to compare gene function across related species. In addition to animal models, TALENs have been used to introduce targeted alterations in plants, like Arabidopsis and several crop species, allowing the incorporation of valuable traits, such as disease and herbicide resistance.
A newer technology CRISPR/Cas9 has become more popular over other genome editing tools because of its easiest programmability. However, TALEN provides a valuable option because of its unconstrained target site requirement and high degree of target specificity with less off-target effects.
1. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, et al. 2009. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509-12\
2. Bogdanove AJ, Voytas DF. 2011. TAL effectors: customizable proteins for DNA targeting. Science 333:1843-46
About Author / Additional Info:
I am a research scientist pursuing work on TAL effectors in Xanthomonas oryzae pv oryzae, the casual organism of bacterial leaf blight of rice.
Comments on this article: (0 comments so far)
• Malady Diagnosis and Remedies in Important Fruit Crops
• Bacterial Antifreeze Proteins and Its Applications
• Proteomics - A Promising Approach of Biotechnology
• What is DNA Dating? (or Gene Pairing Technique)
Latest Articles in "Biotech-Research" category:
• Human Longevity: A Revolution in Biotechnology and Nanotechnology.
• Nanoparticles as Delivery Device For Gene Therapy
• Biotechnology as a Tool in Medicine: Focus on Artemisinin
• Tissue Cells and Skin Cells Reprogrammed Into Embryonic Stem Cells:-
• Polymerase Chain Reaction (or PCR) - Technique For Amplifying DNA
• Treatment of Heart Disease With Stem Cells
• Biological Activities and Bioassays
• DNA Sequencing: Maxam Gilbert Method
• PCR Aspects and its Future | PCR versus Cloning
• Plasmid as Vectors For Plant Transformation
• Gene Isolation and Characterisation
• Apoptosis and Cancer: A Review
• Extraction of Nucleic Acids (DNA and RNA) From Plant Tissues
• Stem Cells From Bone Marrow and Vein Leftovers Can Heal Damaged Hearts
• Gene Transfer Techniques: Biolistics, Bacterial and Viral Transformation
• Breast Cancer: Cactus For Womens Life
• Mtt Assay: Assess The Viability Of Cell In Culture
• Medicinal Plants: Source Of Medicine
• Biotechnology Impact on Alzheimer's Disease
Important Disclaimer: All articles on this website are for general information only and is not a professional or experts advice. We do not own any responsibility for correctness or authenticity of the information presented in this article, or any loss or injury resulting from it. We do not endorse these articles, we are neither affiliated with the authors of these articles nor responsible for their content. Please see our disclaimer section for complete terms.
Copyright © 2010 biotecharticles.com - Do not copy articles from this website.
ARTICLE CATEGORIES :
| Disclaimer/Privacy/TOS | Submission Guidelines | Contact Us