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) (Jasin, 1996). DSBs can stimulate recombination efficiency several thousand-fold, approaching gene targeting frequencies as high as 50% (Alwin et al., 2005; Lombardo et al., 2007; Maeder et al., 2008; Moehle et al., 2007; Porteus and Baltimore, 2003; Urnov et al., 2005). Early experiments utilized highly specific homing endonucleases, enzymes that bind and cleave extended DNA sequences, to introduce DSBs into specific genomic loci (Jasin, 1996). To date, however, the use of homing endonucleases to enhance gene targeting has not been demonstrated at an actual endogenous gene locus.

Zinc finger nucleases (ZFNs) provide an alternative to homing endonucleases for introducing site-specific DSBs. ZFNs consist of a DNA-binding zinc finger domain (composed of either three or four fingers) covalently linked to the non-specific DNA cleavage domain of the bacterial FokI restriction endonuclease (Figure 2, left panel) (Carroll, 2004; Huang et al., 1996; Kim et al., 1996; Kim et al., 1997; Smith et al., 1999). 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) (Bibikova et al., 2001; Smith et al., 2000). Dimerization of ZFNs is mediated by the FokI cleavage domain (Bitinaite et al., 1998; Li et al., 1992; Wah et al., 1998) which cleaves within a five or six base pair "spacer" sequence that separates the two inverted "half sites" (Figure 2, right panel) (Bibikova et al., 2001; Porteus et al., 2003; Smith et al., 2000; Urnov et al., 2005). Importantly, because the DNA-binding specificities of zinc finger domains can in principle be re-engineered using various methods (Beerli and Barbas, 2002; Hirsh and Joung, 2004; Jamieson et al., 2003; Lee et al., 2003; Pabo et al., 2001), customized ZFNs can theoretically be constructed to target nearly any gene sequence.

Recent work has shown that ZFNs can be used to direct gene targeting or mutagenic non-homologous end-joining (NHEJ) events to specific endogenous loci and/or integrated reporter genes in insect, plant, and human cells. ZFNs can stimulate recombination in plant (Wright et al., 2005) or human cells (Alwin et al., 2005; Porteus and Baltimore, 2003) between two reciprocally defective copies of a reporter gene. ZFN-mediated gene targeting has been used to effect alteration of endogenous genes in human cells (SCID-associated mutations in the IL2R gene (Lombardo et al., 2007; Moehle et al., 2007; Urnov et al., 2005), and promoter-mutations in the VEGF-A gene (Maeder et al., 2008)) and mutation of endogenous genes in Drosophila (Beumer et al., 2006; Bibikova et al., 2003; Bibikova et al., 2002). ZFNs have also been used to induce highly efficient mutagenesis of four endogenous human genes (CCR5 (Lombardo et al., 2007; Perez et al., 2008), DHFR (Santiago et al., 2008), CFTR and HoxB13 (Maeder et al., 2008)), three endogenous zebrafish genes (Doyon et al., 2008; Meng et al., 2008), and one endogenous plant gene (SuRA in tobacco (Maeder et al., 2008)) by error-prone NHEJ. Collectively, these studies suggest that ZFNs will be immediately useful as research tools and, in the longer term, as therapeutic reagents to manipulate the sequence of any endogenous gene.

Zinc Finger Engineering

Widespread testing and application of the ZFN-mediated gene targeting depends upon the ability of the typical scientific researcher to rapidly construct engineered zinc finger domains. In addition, given that the ranges of desirable ZFN affinity and specificity needed to minimize ZFN cytotoxicity remain poorly understood, for any given target DNA site it will be important to obtain multiple zinc finger domains with various affinities and specificities for testing in cells. Therefore, an ideal method for generating multi-finger proteins would provide a user-friendly approach for generating a series of candidate proteins with a range of affinities and specificities for each target DNA site of interest.

The various zinc finger engineering strategies that have been described to date can be broadly grouped into three categories: modular assembly methods, combinatorial selection-based approaches, and the proprietary platform of the company Sangamo Biosciences. We discuss the modular assembly and combinatorial selection methods below in greater detail. However, we note that although the Sangamo platform has been used successfully to generate ZFN pairs for three different endogenous human genes (Lombardo et al., 2007; Moehle et al., 2007; Perez et al., 2008; Santiago et al., 2008; Urnov et al., 2005), it currently remains a "black box" that is inaccessible to most academic researchers. At present, the overall efficacy rate and methodologic details of the Sangamo platform remain largely unknown. In addition, a large archive of zinc finger domains which is apparently needed to practice the method (Doyon et al., 2008) is not publicly available.

Modular assembly of zinc finger domains.
Various reports in the literature have described rapid "modular assembly" methods in which pre-existing finger "modules" with known, optimized specificities are linked together to create a multi-finger domain. This strategy can use modules consisting of single fingers (obtained by selection from randomized libraries (Mandell and Barbas, 2006), by rational design (Liu et al., 2002), or from naturally occurring domains (Bae et al., 2003)) to assemble multi-finger domains. The ZFC has made a large collection of 141 previously described finger modules available on a unified plasmid framework that enables them to be rapidly assembled into multi-finger domains (Wright et al., 2006). This collection of reagents is available to academic scientists through Addgene, a non-profit plasmid distribution service (http://www.addgene.org/zfc). In addition, associated web-based software that facilitates the identification of sites that can be targeted using these modules has also been described (Sander et al., 2007).

However, although modular assembly is conceptually appealing in its simplicity, a recent large-scale analysis performed by members of the Consortium has shown that the overall success rate of this approach for making three-finger domains is at most ~24% (Ramirez et al., 2008). This analysis also revealed that failure rates for modular assembly depend upon the sequences of the 3 bp "sub-sites" that compose the overall target site. Higher failure rates were observed for sites that are composed of fewer numbers of GNN sub-sites (where N is any base). Because ZFNs function as dimers, two multi-finger domains must be created for each potential cleavage site. Thus, the success rate for making a pair of three-finger ZFNs is estimated to be no more than ~6% (24% of 24%). These results strongly suggest that researchers who wish to use modular assembly to create three-finger ZFNs should expect that this method will fail to yield a functional ZFN pair for the majority of sites targeted.

Combinatorial selection of engineered multi-finger proteins.
A likely reason for the low efficiency of modular assembly is that it ignores context-dependent effects on DNA-binding that have been previously observed with zinc fingers (Elrod-Erickson et al., 1996; Isalan et al., 1997; Isalan et al., 1998; Wolfe et al., 2001). Various selection-based strategies have been described in the literature in which combinations of fingers that work well together to bind a target site are identified from randomized libraries (Greisman and Pabo, 1997; Hurt et al., 2003; Isalan et al., 2001). These various approaches seek to balance the desire to maximize sequence diversity in the starting randomized libraries and the requirement to keep combinatorial possibilities to a manageable level for selections. Although these methods can yield multi-finger domains that bind with high affinities and specificities (Greisman and Pabo, 1997; Hurt et al., 2003) and that function well in human cells(Cornu et al., 2008; Pruett-Miller et al., 2008), all of them require specialized expertise in the construction of large randomized libraries and in the performance of labor-intensive selections. As such, the practice of these methods has remained restricted to only a small number of labs that possess the required experience and know-how.

Recently, member labs of the Zinc Finger Consortium described a simplified combinatorial-based selection method for making zinc finger arrays which we term OPEN (for Oligomerized Pool ENgineering) (Maeder et al., 2008). OPEN selections utilize an archive of pre-selected zinc finger pools, each containing a collection of fingers targeted to a different three base pair subsite at a defined position within the context of a three-finger protein (Figure 3). Fully enabling OPEN will require the construction of 192 finger pools (64 potential three bp target subsites for each position in a three-finger protein). To date, the ZFC has created pools (each containing a maximum of 95 different fingers) targeted to 66 subsites (48 GNN subsites and 18 TNN subsites (Maeder et al., 2008)). To perform an OPEN selection for a target site, appropriate finger pools from the archive are recombined to create a small library of variants (95³ = 8.6 x 10⁵ members for a three-finger domain). This library is then interrogated using a bacterial two-hybrid (B2H) selection system in which binding of a zinc-finger domain to its cognate site activates expression of selectable marker genes (Hurt et al., 2003; Joung et al., 2000; Maeder et al., 2008).

The Zinc Finger Consortium

The Zinc Finger Consortium continues to work to develop robust, publicly available methods for engineering zinc finger nucleases that function well in various cellular environments. The Consortium intends to make all methods, protocols, software, and reagents they develop available to the academic scientific community.

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