What is TALEN?
Transcription activator like effector nuclease (TALEN) is a powerful tool for genome editing. The chimeric nuclease consists of two parts: coding sequence-specific DNA binding modules and non-specific DNA cleavage domains. By inducing DNA double-strand breaks to stimulate error-prone non-homologous end connections or homologous directed repair at specific gene locations.
TAL effector (TALE) was originally discovered as an invasive strategy for bacterial infection of plants in a plant pathogen called Xanthomonas. These TALEs are injected into plant cells through the bacterial type III secretion system, and regulate transcription by targeting effector-specific gene promoters to promote bacterial colony formation. Due to the sequence-specific binding ability of TALE, researchers have formed a powerful class of tools with specific genome editing function, namely TALEN, by linking FokI nuclease with a segment of artificial TALE.
TALEN Typical Structure
A typical TALEN consists of an N-terminal domain containing a nuclear localization signal, NLS, a central domain containing a typical tandem TALE repeat sequence that recognizes a specific DNA sequence, and a C-terminal domain with FokI endonuclease function.Specific DNA_sequence lengths recognized by different types of TALEN elements differ greatly.In general, the length of specific DNA sequence recognized by natural TALEN elements is generally 17-18 bp, while that recognized by artificial TALEN elements is generally 14-20 bp.
TALEN Technical Principle
Figure 1. TALEN technical
The principle of TALEN technology is to target and combine TALEN elements to specific DNA sites by the DNA recognition module, then complete the shearing of specific sites under the action of FokI nuclease, and complete the insertion (or inversion), deletion and gene fusion of specific sequences by means of the inherent homologous directed repair (HDR) or non-homologous end joining pathway (NHEJ) repair process in cells. The core principle of TALEN is to achieve three different functions: directing into the nucleus, specific recognition of target site DNA, and cleavage of target site DNA in an orderly manner. The key to TALEN is to complete the specific recognition function of DNA, which is generally divided into two steps.
(1) Construct TAL target recognition module
The DNA-specific recognition unit of TAL is a double amino acid spaced by 32 constant amino acid residues. Binary amino acids correspond to the four nucleotide bases of AGCT: adenine (A) is recognized by NI, thymine (T) by NG, guanine (G) by N, and cytosine (C) by HD. In the experimental operation, we can deduce the duplex amino acid sequence that can specifically recognize this sequence through the DNA sequence of the target site, thus constructing the TAL target recognition module.
(2) Target recognition module recognition and expression of TAL
We need to connect a pair of TAL target recognition modules constructed according to the target DNA sequence in the previous step with the nuclear localization sequence at the N-terminal and FokI enzyme at the C-terminal to obtain a complete TALEN element. For one thing, we can adopt the eukaryotic expression vector system specially used to construct TALEN, clone a pair of specific TAL target recognition modules into the vector, and then introduce them into cells by transfection and other ways.
Application of TALEN
Since 2011, TALE and TALEN technologies have been rapidly applied in many species, of which zebrafish is the most prominent model animal. The application of TALEN technology has also been extended to more species and has been rapidly developed and tested. Some researchers used this technology to successfully obtain mutants expressing surface antigen H-2K (k) and hygromycin resistance protein, inserting or deleting mutants respectively, and then using magnetic separation and hygromycin treatment for cell separation, respectively. The results showed that the target cells were successfully isolated from cells greatly enriched with gene mutations, indicating that this technology can effectively enrich mutants. Therefore, they recommended that TALEN technology be more widely used in biomedical research, thereby greatly facilitating the efficiency of nucleic acid recombination. Researchers have also used this technology to genomically modify HEK293T cells and to mutate genes in model animals such as human induced pluripotent stem cells (iPS), Drosophila melanogaster, zebrafish, and Xenopus laevis, and it only takes 1 week to directly complete the evaluation in mammalian cells. Compared with the traditional yeast-based evaluation method, this method is more efficient, and the application of TALENS technology in various model organisms will provide a more convenient and effective method. In addition, TALEN was used to successfully knock out the proto-oncogenes of E6 and E7 proteins in human HPV-positive cell lines, thereby promoting cell apoptosis, inhibiting cell growth, reducing the possibility of tumorigenesis, and also enabling functional repair of p53 and RB genes. Thus, the TALEN-mediated genome editing of human cells will become a pillar technology in human biology and disease research. This new technology of gene editing can remove genes efficiently and quickly, and bring about the effect of efficient directional modification for the construction of model animals, gene therapy and modification, and cultivation of new varieties.
TALEN has undoubtedly made breakthroughs in the field of gene editing since its application. In terms of precise gene modification, the application of TALEN makes it possible to study some complex gene functions, and TALEN-mediated gene editing is more effective and fast than traditional gene targeting schemes. At present, TALEN technology has been successful in many species, and its high efficiency has been recognized by the majority of scientific researchers. The only disadvantage of TALEN compared with ZFN is that its expression vector is larger, which may reduce the efficiency of plasmid transfection to some extent. Therefore, improving the introduction of TALEN is an important factor to improve the efficiency of TALEN cleavage.