Zinc-finger nuclease
Directed evolution has been employed to generate a FokI variant with enhanced cleavage activity that the authors dubbed "Sharkey".
[7] Structure-based design has also been employed to improve the cleavage specificity of FokI by modifying the dimerization interface so that only the intended heterodimeric species are active.
[8][9][10][11] Zinc finger nucleases are useful to manipulate the genomes of many plants and animals including arabidopsis,[12][13] tobacco,[14][15] soybean,[16] corn,[17] Drosophila melanogaster,[18] C. elegans,[19] Platynereis dumerilii,[20] sea urchin,[21] silkworm,[22] zebrafish,[23] frogs,[24] mice,[25] rats,[26] rabbits,[27] pigs,[28] cattle,[29] and various types of mammalian cells.
ZFNs 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).
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.
[34] 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.
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.
They suggested that the ZFN 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.
ZFNs 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.
Custom-designed ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc-finger 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.
Such off-target cleavage may lead to the production of enough double-strand breaks to overwhelm the repair machinery and, as a consequence, yield chromosomal rearrangements and/or cell death.
[39][40] The ability to precisely manipulate the genomes of plants and animals has numerous applications in basic research, agriculture, and human therapeutics.
Using ZFNs to modify endogenous genes has traditionally been a difficult task due mainly to the challenge of generating zinc finger domains that target the desired sequence with sufficient specificity.
Improved methods of engineering zinc finger domains and the availability of ZFNs from a commercial supplier now put this technology in the hands of increasing numbers of researchers.
[49] These SSBs undergo the same cellular mechanisms for DNA that ZFNs exploit, but they show a significantly reduced frequency of mutagenic NHEJ repairs at their target nicking site.
ZFNickases can induce targeted HR in cultured human and livestock cells, although at lower levels than corresponding ZFNs from which they were derived because nicks can be repaired without genetic alteration.
