difference between these engineered nucleases is in their dna recognition peptide. zfns rely on cys2-his2 zinc fingers and talen constructs on tales. both of these dna recognizing peptide domains have the characteristic that they are naturally found in combinations in their proteins. cys2-his2 zinc fingers typically happen in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins such as transcription factors. each finger of the zinc finger domain is completely independent and the binding capacity of one finger is impacted by its neighbor. tales on the other hand are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs. because both zinc fingers and tales happen in repeated patterns, different combinations can be tried to create a wide variety of sequence specificities. zinc fingers have been more established in these terms and approaches such as modular assembly (where zinc fingers correlated with a triplet sequence are attached in a row to cover the required sequence), open (low-stringency selection of peptide domains vs. triplet nucleotides followed by high-stringency selections of peptide combination vs. the final target in bacterial systems), and bacterial one-hybrid screening of zinc finger libraries among other methods have been used to make site specific nucleases. zinc finger nucleases are research and development tools that have already been used to modify a range of genomes, in particular by the laboratories in the zinc finger consortium. the us company sangamo biosciences uses zinc finger nucleases to carry out research into the genetic engineering of stem cells and the modification of immune cells for therapeutic purposes. modified t lymphocytes are currently undergoing phase i clinical trials to treat a type of brain tumor (glioblastoma) and in the fight against aids. talen general overview of the talen process transcription activator-like effector nucleases (talens) are specific dna-binding proteins that feature an array of 33 or 34-amino acid repeats. talens are artificial restriction enzymes designed by fusing the dna cutting domain of a nuclease to tale domains, which can be tailored to specifically recognize a unique dna sequence. these fusion proteins serve as readily targetable "dna scissors" for gene editing applications that enable to perform targeted genome modifications such as sequence insertion, deletion, repair and replacement in living cells. the dna binding domains, which can be designed to bind any desired dna sequence, comes from tal effectors, dna-binding proteins excreted by plant pathogenic xanthomanos app. tal effectors consists of repeated domains, each of which contains a highly conserved sequence of 34 amino acids, and recognize a single dna nucleotide within the target site. the nuclease can create double strand breaks at the target site that can be repaired by error-prone non-homologous end-joining (nhej), resulting in gene disruptions through the introduction of small insertions or deletions. each repeat is conserved, with the exception of the so-called repeat variable di-residues (rvds) at amino acid positions 12 and 13. the rvds determine the dna sequence to which the tale will bind. this simple one-to-one correspondence between the tale repeats and the corresponding dna sequence makes the process of assembling repeat arrays to recognize novel dna sequences straightforward. these tales can be fused to the catalytic domain from a dna nuclease, foki, to generate a transcription activator-like effector nuclease (talen). the resultant talen constructs combine specificity and activity, effectively generating engineered sequence-specific nucleases that bind and cleave dna sequences only at pre-selected sites. the talen target recognition system is based on an easy-to-predict code. tal nucleases are specific to their target due in part to the length of their 30+ base pairs binding site. talen can be performed within a 6 base pairs range of any single nucleotide in the entire genome. talen constructs are used in a similar way to designed zinc finger nucleases, and have three advantages in targeted mutagenesis: (1) dna binding specificity is higher, (2) off-target effects are lower, and (3) construction of dna-binding domains is easier. crispr crisprs (clustered regularly interspaced short palindromic repeats) are genetic elements that bacteria use as a kind of acquired immunity to protect against viruses. they consist of short sequences that originate from viral genomes and have been incorporated into the bacterial genome. cas (crispr associated proteins) process these sequences and cut matching viral dna sequences. by introducing plasmids containing cas genes and specifically constructed crisprs into eukaryotic cells, the eukaryotic genome can be cut at any desired position. editing by nucleobase modification (base editing) one of the earliest methods of efficiently editing nucleic acids employs nucleobase modifying enzymes directed by nucleic acid guide sequences was first described in the 1990s and has seen resurgence more recently. this method has the advantage that it does not require breaking the genomic dna strands, and thus avoids the random insertion and deletions associated with dna strand breakage. it is only appropriate for precise editing requiring single nucleotide changes and has found to be highly efficient for this type of editing. arcut arcut stands for artificial restriction dna cutter, it is a technique developed by komiyama. this method uses pseudo-complementary peptide nucleic acid (pcpna), for identifying cleavage site within the chromosome. once pcpna specifies the site, excision is carried out by cerium (ce) and edta (chemical mixture), which performs the splicing function. precision and efficiency of engineered nucleases meganucleases method of gene editing is the least efficient of the methods mentioned above. due to the nature of its dna-binding element and the cleaving element, it is limited to recognizing one potential target every 1,000 nucleotides. zfn was developed to overcome the limitations of meganuclease. the number of possible targets zfn can recognized was increased to one in every 140 nucleotides. however, both methods are unpredictable because of their dna-binding elements affecting each other. as a result, high degrees of expertise and lengthy and costly validations processes are required. tale nucleases being the most precise and specific method yields a higher efficiency than the previous two methods. it achieves such efficiency because the dna-binding element consists of an array of tale subunits, each of them having the capability of recognizing a specific dna nucleotide chain independent from others, resulting in a higher number of target sites with high precision. new tale nucleases take about one week and a few hundred dollars to create, with specific expertise in molecular biology and protein engineering. crispr nucleases have a slightly lower precision when compared to the tale nucleases. this is caused by the need of having a specific nucleotide at one end in order to produce the guide rna that crispr uses to repair the double-strand break it induces. it has been shown to be the quickest and cheapest method, only costing less than two hundred dollars and a few days of time. crispr also requires the least amount of expertise in molecular biology as the design lays in the guide rna instead of the proteins. one major advantage that crispr has over the zfn and talen methods is that it can be directed to target different dna sequences using its ~80nt crispr sgrnas, while both zfn and talen methods required construction and testing of the proteins created for targeting each dna sequence. because off-target activity of an active nuclease would have potentially dangerous consequences at the genetic and organismal levels, the precision of meganucleases, zfns, crispr, and talen-based fusions has been an active area of research. while variable figures have been reported, zfns tend to have more cytotoxicity than talen methods or rna-guided nucleases, while talen and rna-guided approaches tend to have the greatest efficiency and fewer off-target effects. based on the maximum theoretical distance between dna binding and nuclease activity, talen approaches result in the greatest precision. multiplex automated genomic engineering (mage) synthetic dna is repeatedly introduced at multiple targeted areas of the chromosome and/or loci and then is replicated producing cells with/without mutations. the methods for scientists and researchers wanting to study genomic diversity and all possible associated phenotypes were very slow, expensive, and inefficient. prior to this new revolution, researchers would have to do single-gene manipulations and tweak the genome one little section at a time, observe the phenotype, and start the process over with a different single-gene manipulation. therefore, researchers at the wyss institute at harvard university designed the mage, a powerful technology that improves the process of in vivo genome editing. it allows for quick and efficient manipulations of a genome, all happening in a machine small enough to put on top of a small kitchen table. those mutations combine with the variation that naturally occurs during cell mitosis creating billions of cellular mutations. chemically combined, synthetic single-stranded dna (ssdna) and a pool of oligionucleotides are introduced at targeted areas of the cell thereby creating genetic modifications. the cyclical process involves transformation of ssdna (by electroporation) followed by outgrowth, during which bacteriophage homologous recombination proteins mediate annealing of ssdnas to their genomic targets. experiments targeting selective phenotypic markers are screened and identified by plating the cells on differential medias. each cycle ultimately takes 2.5 hours to process, with additional time required to grow isogenic cultures and characterize mutations. by iteratively introducing libraries of mutagenic ssdnas targeting multiple sites, mage can generate combinatorial genetic diversity in a cell population. there can be up to 50 genome edits, from single nucleotide base pairs to whole genome or gene networks simultaneously with results in a matter of days. mage experiments can be divided into three classes, characterized by varying degrees of scale and complexity: (i) many target sites, single genetic mutations; (ii) single target site, many genetic mutations; and (iii) many target sites, many genetic mutations. an example of class three was reflected in 2009, where church and colleagues were able to program escherichia coli to produce five times the normal amount of lycopene, an antioxidant normally found in tomato seeds and linked to anti-cancer properties. they applied mage to optimize the 1-deoxy-d-xylulose-5-phosphate (dxp) metabolic pathway in escherichia coli to overproduce isoprenoid lycopene. it took them about 3 days and just over $1,000 in materials. the ease, speed, and cost efficiency in which mage can alter genomes can transform how industries approach the manufacturing and production of important compounds in the bioengineering, bioenergy, biomedical engineering, synthetic biology, pharmaceutical, agricultural, and chemical industries. applications plants, animals and human genes that are successfully targeted using zfn, which demonstrates the generality of this approach as of 2012 efficient genome editing had been developed for a wide range of experimental systems ranging from plants to animals, often beyond clinical interest, and was becoming a standard experimental strategy in research labs. the recent generation of rat, zebrafish, maize and tobacco zfn-mediated mutants and the improvements in talen-based approaches testify to the significance of the methods, and the list is expanding rapidly. genome editing with engineered nucleases will likely contribute to many fields of life sciences from studying gene functions in plants and animals to gene therapy in humans. for instance, the field of synthetic biology which aims to engineer cells and organisms to perform novel functions, is likely to benefit from the ability of engineered nuclease to add or remove genomic elements and therefore create complex systems. in addition, gene functions can be studied using stem cells with engineered nucleases. listed below are some specific tasks this method can carry out: targeted gene mutation gene therapy creating chromosome rearrangement study gene function with stem cells transgenic animals endogenous gene labeling targeted transgene addition targeted gene modification in animals the combination of recent discoveries in genetic engineering, particularly gene editing and the latest improvement in bovine reproduction technologies (e.g. in vitro embryo culture) allows for genome editing directly in fertilised oocytes using synthetic highly specific endonucleases. rna-guided endonucleases:clustered regularly interspaced short palindromic repeats associated cas9 (crispr/cas9) are a new tool, further increasing the range of methods available. in particular crispr/cas9 engineered endonucleases allows the use of multiple guide rnas for simultaneous knockouts (ko) in one step by cytoplasmic direct injection (cdi) on mammalian zygotes. furthermore, gene editing can be applied to certain types of fish in aquaculture such as atlantic salmon. gene editing in fish is currently experimental, but the possibilities include growth, disease resistance, sterility, controlled reproduction, and colour. selecting for these traits can allow for a more sustainable environment and better welfare for the fish. aquadvantage