Zinc Finger Nuclease - DNA-binding Domain

DNA-binding Domain

The DNA-binding domains of individual ZFNs typically contain between three and six individual zinc finger repeats and can each recognize between 9 and 18 basepairs. If the zinc finger domains are perfectly specific for their intended target site then even a pair of 3-finger ZFNs that recognize a total of 18 basepairs can theoretically target a single locus in a mammalian genome.

Various strategies have been developed to engineer Cys₂His₂ zinc fingers to bind desired sequences. These include both "modular assembly" and selection strategies that employ either phage display or cellular selection systems.

The most straightforward method to generate new zinc-finger arrays is to combine smaller zinc-finger "modules" of known specificity. The most common modular assembly process involves combining three separate zinc fingers that can each recognize a 3 basepair DNA sequence to generate a 3-finger array that can recognize a 9 basepair target site. Other procedures can utilize either 1-finger or 2-finger modules to generate zinc-finger arrays with six or more individual zinc fingers. The main drawback with this procedure is the specificities of individual zinc fingers can overlap and can depend on the context of the surrounding zinc fingers and DNA. Without methods to account for this "context dependence", the standard modular assembly procedure often fails unless it is used to recognize sequences of the form (GNN)_N.

Numerous selection methods have been used to generate zinc-finger arrays capable of targeting desired sequences. Initial selection efforts utilized phage display to select proteins that bound a given DNA target from a large pool of partially randomized zinc-finger arrays. More recent efforts have utilized yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells. A promising new method to select novel zinc-finger arrays utilizes a bacterial two-hybrid system and has been dubbed "OPEN" by its creators. This system combines pre-selected pools of individual zinc fingers that were each selected to bind a given triplet and then utilizes a second round of selection to obtain 3-finger arrays capable of binding a desired 9-bp sequence. This system was developed by the Zinc-Finger Consortium as an alternative to commercial sources of engineered zinc-finger arrays.

(see: Zinc finger chimera for more info on zinc finger selection techniques)