Zinc finger nuclease

Zinkfingernukleasen ( ZFN ) are man-made restriction enzymes. They contain a zinc finger domain that binds to DNA and a nuclease which cuts the DNA. The zinc finger domain can be built so that it recognizes a specific DNA sequence. This means that you can cut a complex genome at a specific site with ZFN, enabling a targeted incorporation of foreign DNA.

Nuclease

Zinkfingernukleasen usually contain the non-specific nuclease cutting of the type IIS restriction enzyme FokI. This restriction domain is active only when it is present dimerizes, which is why two different built ZFN monomers are required to cut the DNA target sequence. For standard ZFN the domain restriction is bound via its N-terminal end of the DNA-binding zinc finger domain. So that the nuclease dimerize and can cut the two different monomers must ZFN binding to the two different strands of the DNA, and their C-termini must have a certain distance from each other.

A plurality of different protein engineering techniques can be used to enhance the enzyme activity on the one hand and on the other hand, affinity and specificity of the zinc finger domain. For example, " directed evolution " is applied to generate FokI variants that have increased nuclease. In addition, structural design was used to generate by means of exchange of charged amino acids in the Dimerisierungsinterface nuclease called " obligate heterodimer " FokI variants that have a significantly increased specificity of restriction.

DNA - binding domain

The DNA - binding domain typically contains between three and six different zinc finger motifs, each individual zinc finger motif recognizes 3 bp. Bind the zinc finger domains perfectly to their recognition sequence, so rich three zinc fingers per ZFN monomer, in order to detect a given locus in a complex genome specific. Several different strategies have been developed to obtain Cys2His2 zinc fingers that bind to the desired sequences. These methods include both the modular assembly (see below) as well as selection strategies such as phage display, yeast- 1- hybrid systems, one- hybrid Bacterial system Bacterial two-hybrid system or cellular selection systems.

The easiest way to generate new zinc finger array is the combination of zinc fingers with known specificity. The most widely used process is the modular assembly combining three different zinc fingers that recognize each 3 bp to a new zinc finger array that detects 9 bp. The main disadvantage of this method is that the specificity of a zinc finger may change depending on the adjacent zinc fingers in the array, which is why " context- dependent" selection strategies usually produce zinc finger arrays with a higher specificity.

Applications

Meanwhile Zinkfingernukleasen were used for the targeted modification of several plant and animal genomes, including Arabidopsis, tobacco, soybeans, corn, fruit fly, nematode, sea urchins, silkworm, zebrafish, frog, mouse, rat, rabbit, pork, beef and various types of mammalian cells. In addition, ZFN were applied in vivo in a mouse model of hemophilia, as well as in an ongoing clinical study with the aim to kill the CCR5 gene in autologous CD4 T cells as a possible therapeutic approach for the treatment of HIV infection or AIDS.