<unk> s are useful to manipulate the genomes of many plants and animals including arabidopsis, tobacco, soybean, corn, Drosophila melanogaster, C. elegans, Platynereis dumerilii, sea urchin, silkworm, zebrafish, frogs, mice, rats, rabbits, pigs, cattle, and various types of mammalian cells.<unk> s have also been used in a mouse model of haemophilia and a clinical trial found CD4+ human T-cells with the CCR5 gene disrupted by<unk> s to be safe as a potential treatment for HIV/AIDS.<unk> s are also used to create a new generation of genetic disease models called isogenic human disease models. Disabling an allele<unk> s can be used to disable dominant mutations in heterozygous individuals by producing double-strand breaks (DSBs) in the DNA (see Genetic recombination) in the mutant allele, which will, in the absence of a homologous template, be repaired by non-homologous end-joining (NHEJ). NHEJ repairs DSBs by joining the two ends together and usually produces no mutations, provided that the cut is clean and uncomplicated. In some instances, however, the repair is imperfect, resulting in deletion or insertion of base-pairs, producing frame-shift and preventing the production of the harmful protein. Multiple pairs of<unk> s can also be used to completely remove entire large segments of genomic sequence. To monitor the editing activity, a PCR of the target area amplifies both alleles and, if one contains an insertion, deletion, or mutation, it results in a heteroduplex single-strand bubble that cleavage assays can easily detect.<unk> s have also been used to modify disease-causing alleles in triplet repeat disorders. Expanded CAG/CTG repeat tracts are the genetic basis for more than a dozen inherited neurological disorders including Huntington's disease, myotonic dystrophy, and several spinocerebellar ataxias. It has been demonstrated in human cells that<unk> s can direct double-strand breaks (DSBs) to CAG repeats and shrink the repeat from long pathological lengths to short, less toxic lengths. Recently, a group of researchers have successfully applied the<unk>  technology to genetically modify the gol pigment gene and the ntl gene in zebrafish embryo. Specific<unk><unk>  motifs were engineered to recognize distinct DNA sequences. The<unk> -encoding mRNA was injected into one-cell embryos and a high percentage of animals carried the desired mutations and phenotypes. Their research work demonstrated that<unk> s can specifically and efficiently create heritable mutant alleles at loci of interest in the germ line, and<unk> -induced alleles can be propagated in subsequent generations. Similar research of using<unk> s to create specific mutations in zebrafish embryo has also been carried out by other research groups. The kdr gene in zebra fish encodes for the vascular endothelial growth factor-2 receptor. Mutagenic lesions at this target site was induced using<unk>  technique by a group of researchers in US. They suggested that the<unk>  technique allows straightforward generation of a targeted allelic series of mutants; it does not rely on the existence of species-specific embryonic stem cell lines and is applicable to other vertebrates, especially those whose embryos are easily available; finally, it is also feasible to achieve targeted knock-ins in zebrafish, therefore it is possible to create human disease models that are heretofore inaccessible. Allele editing<unk> s are also used to rewrite the sequence of an allele by invoking the homologous recombination (HR) machinery to repair the DSB using the supplied DNA fragment as a template. The HR machinery searches for homology between the damaged chromosome and the extra-chromosomal fragment and copies the sequence of the fragment between the two broken ends of the chromosome, regardless of whether the fragment contains the original sequence. If the subject is homozygous for the target allele, the efficiency of the technique is reduced since the undamaged copy of the allele may be used as a template for repair instead of the supplied fragment. Gene therapy The success of gene therapy depends on the efficient insertion of therapeutic genes at an appropriate chromosomal target site within the human genome, without causing cell injury, oncogenic mutations, or an immune response. The construction of plasmid vectors is simple and straightforward. Custom-designed<unk> s that combine the non-specific cleavage domain (N) of FokI endonuclease with<unk><unk>  proteins (ZFPs) offer a general way to deliver a site-specific DSB to the genome, and stimulate local homologous recombination by several orders of magnitude. This makes targeted gene correction or genome editing a viable option in human cells. Since<unk> -encoding plasmids could be used to transiently express<unk> s to target a DSB to a specific gene locus in human cells, they offer an excellent way for targeted delivery of the therapeutic genes to a pre-selected chromosomal site. The<unk> -encoding plasmid-based approach has the potential to circumvent all the problems associated with the viral delivery of therapeutic genes. The first therapeutic applications of<unk> s are likely to involve ex vivo therapy using a patients own stem cells. After editing the stem cell genome, the cells could be expanded in culture and reinserted into the patient to produce differentiated cells with corrected functions. Initial targets likely include the causes of monogenic diseases, such as the IL2Rγ gene and the β-globin gene for gene correction and CCR5 gene for mutagenesis and disablement.
Zinc finger nuclease
Signal peptide peptidase
Transfer factor
Dispase
Core binding factor
Tenase
Transfersome
Thymidine phosphorylase