Restriction enzymes are enzymes that cut DNA strands at a specific sequence. Transcription activator-like effectors (TALEs) can be quickly engineered to bind practically any desired DNA sequence. By combining such an engineered TALE with a DNA cleavage domain (which cuts DNA strands), one can engineer restriction enzymes that will specifically cut any desired DNA sequence. When these restriction enzymes are introduced into cells, they can be used for gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases. Alongside zinc finger nucleases and Cas9 proteins, TALEN is becoming a prominent tool in the field of genome editing.
TAL effector DNA-binding domain
TAL effectors are proteins that are secreted by Xanthomonas bacteria. The DNA binding domain contains a repeated highly conserved 33–34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the Repeat Variable Diresidue (RVD), are highly variable and show a strong correlation with specific nucleotide recognition. This relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA-binding domains by selecting a combination of repeat segments containing the appropriate RVDs.
DNA cleavage domain
The non-specific DNA cleavage domain from the end of the FokI endonuclease can be used to construct hybrid nucleases that are active in many different cell types. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity.
Figure 1. Workflow of genome editing of Your Favorite Gene (YFG) using TALEN technology. The targent sequence is identified, a corresponding TALEN sequence is engineered and inserted into a plasmid. The plasmid is inserted into the target cell where it is translated to produce the functional TALEN, which enters the nucleus and binds and cleaves the target sequence. Depending on the application, this can be used to introduce an error (to knock out a target gene) or to introduce a new DNA sequence into the target gene.
The simple relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for the efficient engineering of proteins. Once the TALEN constructs have been assembled, they are inserted into plasmids; the target cells are then transfected with the plasmids, and the gene products are expressed and enter the nucleus to access the genome. Alternatively, TALEN constructs can be delivered to the cells as mRNAs, which removes the possibility of genomic integration of the TALEN-expressing protein. Using an mRNA vector can also dramatically increase the level of homology directed repair (HDR) and the success of introgression during gene editing.
Video 1. Gene-editing nucleases. In this video, Nature Methods technology editor Monya Baker explains how gene-editing nucleases work and why they were chosen as Nature Methods 'Method of the Year' for 2011.
Gene Therapy Application
TALEN technology has been used for instance to efficiently engineer stably modified human embryonic stem cell and induced pluripotent stem cell (IPSCs) clones and human erythroid cell lines. The technology has also been utilized experimentally to correct the genetic errors that underlie disease. For example, it has been used in vitro to correct the genetic defects that cause disorders such as sickle cell disease, xeroderma pigmentosum, and epidermolysis bullosa. It was also shown that TALEN technology can be used as tools to harness the immune system to fight cancers. In theory, 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. In reality, however, the in situ application of TALEN® technology is currently limited by the lack of an efficient delivery mechanism, unknown immunogenic factors, and uncertainty in the specificity of TALEN binding. Another emerging application of TALEN® technology 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.