Abstract
Recent work has shown that it is possible to target regulatory elements to DNA sequences of an investigator's choosing, increasing the armamentarium for probing gene function. In this review, we discuss the development and use of designer zinc finger proteins (ZFPs) as sequence specific tools. While the main focus of this review is to discuss the attachment of the FokI nuclease to ZFPs and the ability of the resulting fusion protein (termed zinc finger nucleases (ZFNs)) to genomically manipulate a gene of interest, we will also cover the utility of other functional domains, such as transcriptional activators and repressors, and highlight how these are being used as discovery and therapeutic tools.
Highlights
The expression repertoire of all the genes in any given cell is governed by transcriptional activators and repressors that bind to specific sites in the genome
Designer zinc finger protein (ZFP) are already in clinical trials, and a multitude of others are in development
It is clear that some regions of the genome are more targetable than others by ZFPs; the reason for this is not completely clear, and until this is resolved, progress will remain somewhat impeded
Summary
The expression repertoire of all the genes in any given cell is governed by transcriptional activators and repressors that bind to specific sites in the genome. Each zinc finger binds three nucleotides, with the entire Zif268 polypeptide binding a nine base pair (bp) GCG-TGG-GCG DNA motif Such a modular design immediately suggested the possibility for combining zinc fingers with distinct triplet recognition motifs, to create proteins that could potentially recognize any DNA sequence. While modular assembly provided examples of successfully applied ZFPs, it was observed that a high failure rate occurred with this approach [7] Because this deficiency is likely due to the influence that neighboring zinc-fingers have on the sequence specificity of a given zinc finger [8,9,10,11], selection-guided assemblies were developed [12,13,14,15,16,17]. The resulting synthetic zinc finger trimer can display high affinity and
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