Meganucleases are enzymes that are both endonuclease and deoxyribonuclease. They are characterized by a big receptive site. This receptive site has double-stranded DNA. Consequently, the site generally occurs once in a particular genome. A good example is the eighteen base pair sequence identified by the I-Sel meganuclease.
This requires a genome that is greater than the size of the human genome (this genome is found by chance, therefore, it is not always available). Meganuclease is known to be a good restriction enzyme that is specific in action.
Meganuclease as Tools for Genome Editing
Genome editing has a wide frame in recent years. Recent findings explained the process of genome editing. Genome editing methods are the techniques used where the DNA sequence is altered by omission, mRNA processing, and the post-transcriptional alterations to the result in tailored gene expressions. This, consequently, leads to the effective functions of proteins.
Meganuclease has advanced from just targeting genes to transgenic approaches. Meganuclease has a characteristic of being specific in actions which gives them a high degree of accuracy and precision. They are less toxic to cells. Meganucleases were identified and named in the 1990s and recent studies have shown that they are effective tools for genome editing and gene engineering.
This is so because they are efficacious in the generation of mutations, catalyze reading frames, and they also support homologous recombination. The meganuclease-aided genetic recombination that could be displayed was restricted by the inventory of the available meganuclease.
Each meganuclease can permit little variations in its receptive site even though there are a good number of them in nature. It is a difficult thing to get a meganuclease that can cut a particular gene at a given location. The attention of scientists was drawn to the engineering of new meganuclease with the sole purpose of targeting a particular receptive site.
They create avenues for the elimination of bad genetic material and they also help to repair damaged genes.
Genome editing is a way of changing the DNA sequence of plants, animals, and bacteria in other to amplify a specific property. It makes use of DNA editing in explaining changes characterized by the change in physical traits.
Biotechnology detailed the information needed to edit DNA and thus, strengthen the downstream pathway. Since meganuclease is, sometimes, found in the genome, they have the genetic material required to modify DNA. Meganuclease is joined by proteins to produce large variants which include Dmocre and E-Drel.
These variants also create nucleotide receptive site-specific cleavage. This method has two starting steps which are the identification of a cleavage site and after that, the endonuclease will combine the regions.
Processes of Creating Meganucleases
One of the processes of creating meganucleases is the modification of the accuracy of subsisting meganucleases by the introduction of a little amount of variations to the protein sequence. After that, the selection of the functional amino acids on variations of the original receptive site is also viable.
Another process is the exploitation of the property that is very important in meganucleases’ high rate of diversification (it is possible to fuse amino acid domains from different enzymes site). The production of chimeric meganucleases with a different recognition site that has a half-site of amino-acids is made possible by this process.
The fusion of I-DMol and I-Crel (protein domain) created two chimeric meganucleases with this method (E-Drel and DmoCre). It is also possible to maintain a high rate of effectiveness, precision, and accuracy by combining the two methods to increase the possibility of creating a new sequence of enzymes. The collection of protein domains was made from the homodimeric meganuclease I-Crel and other frameworks.
The clustered protein domains are combined to form engineered heterodimers in the laboratory. The integration of the DNA receptive site from TAL (transcription activation-like) effectors into two different types of nucleases is the advance application of meganucleases for genome editing.
The effectiveness of meganuclease can be affected by both chromatin structure and DNA methylation. To apply these enzymes with efficacy, the epigenetic and genetic code context of a sequence under consideration must be thoroughly calculated. Meganuclease can be used to edit all forms of genome be it animal, plant, or bacterial.
A new meganuclease with promising specificity is generated by the computation of the features of protein- DNA sequence (Chevalier et al. 2002). This method allowed the gene coding with residue that results in a great decrease in the sophistication of the cluster of mutations that is yet to be processed.
In other to use I-Crel as a sample for the framework, many groups altered the DNA receptive properties of the protein altered (Seligman et al. 2002; Sussman et al.2004). Gimble and his colleagues modified the DNA binding site of PI-Sel and chose different protein with edited receptor site using a two-hybrid arrangement (Gimble et al.2003)
Improvement of the Meganuclease Specificity
A lot of meganucleases are heterodimers. They are made up of two monomers that are engineered separately. Each of the monomers must be able to identify half of the DNA target (Stoddard. 2011). The heterodimer is the result of the form shown by the two monomers in the same cell.
Consequently, three combinations of meganuclease atoms are produced inside the cell, the two homodimer by-products, and the heterodimer. The homodimer by-product can destroy the specificity of meganuclease by leading to the off-site cleavage. The homodimer by-product can be stopped by putting the specificity in jeopardy with three methods.
The first method is the generation of special heterodimers by modifying the protein-protein between the point of intersection between two monomers to create heterodimerization and the formation of homodimers. The second method is the joining of two monomers into one unit molecule. The third method includes the binding of a meganuclease with a natural DNA-binding domain i.e. the binding of DNA receptor site from TALE (transcription activator-like effectors) to the C- terminus of meganuclease.
The effectiveness of meganuclease could also be affected by the optimization of the chromosome context of the receptor site sequence.
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