Zinc-finger nucleases: emergence of next generation

Methods of modifying the human genome precisely and efficiently hold great promise for revolutionizing the gene therapy arena. One particularly promising technology is based on the homologous recombination (HR) pathway and is known as gene targeting. Until recently, the low frequency of HR in mammalian cells, and the resulting dependence on selection to identify these rare events, has prevented gene targeting from being applied in a therapeutic context. However, recent advances in generating customized zinc-finger nucleases (ZFNs) that can create a DNA double-strand break (DSB) at preselected sites in the human genome have paved the way for HR-based strategies in gene therapy. By introducing a DSB into a target locus of interest, ZFNs stimulate gene targeting by several orders of magnitude through activation of cellular DNA repair pathways. The capability of this technology to achieve gene conversion frequencies of up to 29% in the absence of selection demonstrates its potential power. In this paper we review recent advances in, and upcoming challenges for, this emerging technology and discuss future experimental work that will be needed to bring ZFNs safely into a clinical setting.

Zinc-finger nuclease (ZFN)-mediated genome editing. (a) Architecture and application of ZFNs. A ZFN designed to create a DNA double-strand break (DSB) in the target locus is composed of two monomer subunits. Each subunit encompasses three zinc-fingers (orange, 1-2-3), which recognize 9 base pairs within the full target site, and the FokI endonuclease domain (green). A short linker (grey) connects the two domains. After dimerization the nuclease is activated and cuts the DNA in the spacer sequence, separating the two target half-sites (L) and (R). (b) Gene disruption. A DSB (yellow flash) introduced by the ZFN into a dominant mutant allele (geneA^dn) is repaired by the error-prone nonhomologous end-joining pathway. Deletions and insertions that can occur disrupt the coding sequence (geneA^–) and render the expressed protein nonfunctional. (c) Gene correction. In order to restore a genetic defect directly in the genome (geneA^mut), a targeting vector (donor DNA) encompassing wild-type sequences homologous to the mutant gene (grey areas) is transduced into the target cell. A ZFN-induced DSB stimulates homologous recombination (HR) between the donor DNA and the defective gene (geneA^mut) to generate a corrected locus (geneA^WT). (d) Gene addition. In order to restore the phenotype of a cell harboring a genetic defect (geneA^mut), a partial cDNA flanked by sequences homologous to the mutant gene, is embedded in a targeting vector. A ZFN-induced DSB stimulates HR between the donor DNA and the mutant gene. Expression of the gene is reconstituted (geneA⁺) and remains under the control of the endogenous promoter.

Zinc-finger engineering platforms. (a) "Modular assembly" involves the joining of single zinc-finger domains of known DNA-binding specificities. Large archives of single-finger modules have been created by selection from randomized libraries using phage display. This approach is conceptually simple but neglects positional and context-dependent effects. (b) "Context-sensitive selection" strategies attempt to identify combinations of zinc-fingers that work well together. One particular strategy for performing such a selection (among several described in the literature) is shown in the figure. The first selection step takes into account the relative position of the finger (1, 2, or 3), while the second step factors in the impact of the respective neighbor(s). (c) The "2 + 2 strategy" is a proprietary platform and details are not known. It is likely that the four-finger domains are assembled from pre-existing archives of two-finger units with known DNA-binding specificities, followed by further optimization using an algorithm-based approach.

Various sources for zinc-finger nuclease (ZFN) off-target activity: (a) insufficient specificity of DNA-binding, permitting ZFN binding to unintended DNA sites, (b) ambiguity of the interdomain linker, allowing cleavage at noncanonical spacer lengths (e.g., at a 7-base-pair (bp) spacer instead of the intended 6-bp spacer), (c) cleavage at isolated target half-sites, and (d) cleavage by homodimeric ZFNs. (e) An ideal ZFN architecture consists of an affinity-matured DNA-binding domain, an optimized linker sequence, and a destabilized and asymmetric dimer interface that regulates the FokI cleavage activity.

Reference authors:

Toni Cathomen¹ and J Keith Joung^2,3

1. ¹Institute of Virology (CBF), Charité Medical School, Berlin, Germany
2. ²Molecular Pathology Unit and Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
3. ³Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

Correspondence: Toni Cathomen, Charité Medical School, Institute of Virology (CBF), Hindenburgdamm 27, D-12203 Berlin, Germany. E-mail: toni.cathomen@charite.de