Introduction

Zinc finger nucleases (ZFNs) are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to the cleavage domain of the FokI restriction endonuclease. ZFNs can be used to induce double-stranded breaks (DSBs) in specific DNA sequences and thereby promote site-specific homologous recombination and targeted manipulation of genomic loci in a variety of different cell types. A long-term goal of the Zinc Finger Consortium is to develop ZFNs as broadly applicable and readily accessible molecular tools for performing targeted genetic alterations. The ability to alter the sequence or structure of any gene of interest would be enormously useful for biological research and molecular therapeutics.

Highly efficient, targeted genome manipulation induced by ZFNs

Gene targeting is a method to repair or inactivate any desired gene of interest. Gene targeting strategies use the introduction of a double-stranded break (DSB) into a genomic locus to enhance the efficiency of recombination with an exogenously introduced homologous DNA "repair template" (Figure 1). DSBs can stimulate recombination efficiency several thousand-fold, approaching gene targeting frequencies as high as 50%. ZFNs can be used to introduce targeted DSBs. ZFNs consist of a DNA-binding zinc finger domain (composed of three to six fingers) covalently linked to the non-specific DNA cleavage domain of the bacterial FokI restriction endonuclease (Figure 2, left panel). ZFNs can bind as dimers to their target DNA sites, with each monomer using its zinc finger domain to recognize a "half-site" (Figure 2, right panel). Dimerization of ZFNs is mediated by the FokI cleavage domain which cleaves within a five or six base pair "spacer" sequence that separates the two inverted "half sites" (Figure 2, right panel). Importantly, because the DNA-binding specificities of zinc finger domains can in principle be re-engineered using one of various methods, customized ZFNs can theoretically be constructed to target nearly any gene sequence.

Zinc Finger Engineering

Publicly available methods for engineering zinc finger domains include: (1) Context-dependent Assembly (CoDA), (2) Oligomerized Pool Engineering (OPEN), and (3) Modular Assembly. The Zinc Finger Consortium provides resources and reagents for practicing each of these methods (see the Software Tools, Protocols, Reagents, and ZFC Newsgroup pages).

Members of the Zinc Finger Consortium continue to work to develop robust, publicly available methods for engineering zinc finger nucleases that function well in various cellular environments. All members have committed to making all protocols, software, DNA sequences, and reagents they publish available to the academic scientific community.