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Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.

The anticancer activity of cis -diamminedichloroplatinum( II ) (cisplatin) arises from its ability to damage DNA, with the major adducts formed being intrastrand d(GpG) and d(ApG) crosslinks 1 . These crosslinks bend and unwind the duplex, and the altered structure attracts high-mobility-group domain (HMG) and other proteins 2 . This binding of HMG-domain proteins to cisplatin-modified DNA has been postulated to mediate the antitumour properties of the drug 3 , 4 . Many HMG-domain proteins recognize altered DNA structures such as four-way junctions and cisplatin-modified DNA 5 , but until now the molecular basis for this recognition was unknown. Here we describe mutagenesis, hydroxyl-radical footprinting and X-ray studies that elucidate the structure of a 1:1 cisplatin-modified DNA/HMG-domain complex. Domain A of the structure-specific HMG-domain protein HMG1 binds to the widened minor groove of a 16-base-pair DNA duplex containing a site-specific cis -[Pt(NH 3 ) 2 {d(GpG)-N7(1),-N7(2)}] adduct. The DNA is strongly kinked at a hydrophobic notch created at the platinum–DNA crosslink and protein binding extends exclusively to the 3′ side of the platinated strand. A phenylalanine residue at position 37 intercalates into a hydrophobic notch created at the platinum crosslinked d(GpG) site and binding of the domain is dramatically reduced in a mutant in which alanine is substituted for phenylalanine at this position.

We have developed a bacterial \"two-hybrid\" system that readily allows selection from libraries larger than 108in size. Our bacterial system may be used to study either protein-DNA or protein--protein interactions, and it offers a number of potentially significant advantages over existing yeast-based one-hybrid and two-hybrid methods. We tested our system by selecting zinc finger variants (from a large randomized library) that bind tightly and specifically to desired DNA target sites. Our method allows sequence-specific zinc fingers to be isolated in a single selection step, and thus it should be more rapid than phage display strategies that typically require multiple enrichment/amplification cycles. Given the large library sizes our bacterial-based selection system can handle, this method should provide a powerful tool for identifying and optimizing protein-DNA and protein-protein interactions.

Engineered Cys2His 2zinc finger proteins (ZFPs) can mediate regulation of endogenous gene expression in mammalian cells. Ideally, all zinc fingers in an engineered multifinger protein should be optimized concurrently because cooperative and context-dependent contacts can affect DNA recognition. However, the simultaneous selection of key contacts in even three fingers from fully randomized libraries would require the consideration of$>\\!\\!10^{24}$possible combinations. To address this challenge, we have developed a novel strategy that utilizes directed domain shuffling and rapid cell-based selections. Unlike previously described methods, our strategy is amenable to scale-up and does not sacrifice combinatorial diversity. Using this approach, we have successfully isolated multifinger proteins with improved in vitro and in vivo function. Our results demonstrate that both DNA binding affinity and specificity are important for cellular function and also provide a general approach for optimizing multidomain proteins.

A method is described for selecting DNA-binding proteins that recognize desired sequences. The protocol involves gradually extending a new zinc finger protein across the desired 9- or 10-base pair target site, adding and optimizing one finger at a time. This procedure was tested with a TATA box, a p53 binding site, and a nuclear receptor element, and proteins were obtained that bind with nanomolar dissociation constants and discriminate effectively (greater than 20,000-fold) against nonspecific DNA. This strategy may provide important information about protein-DNA recognition as well as powerful tools for biomedical research.

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics.

Structure-based design was used to link zinc finger peptides and make poly-finger proteins that have dramatically enhanced affinity and specificity. Our studies focused on a fusion in which the three-finger Zif268 peptide was linked to a designed three-finger peptide (designated ``NRE'') that specifically recognizes a nuclear hormone response element. Gel shift assays indicate that this six-finger peptide, 268//NRE, binds to a composite 18-bp DNA site with a dissociation constant in the femtomolar range. We find that the slightly longer linkers used in this fusion protein provide a dramatic improvement in DNA-binding affinity, working much better than the canonical ``TGEKP'' linkers that have been used in previous studies. Tissue culture transfection experiments also show that the 268//NRE peptide is an extremely effective repressor, giving 72-fold repression when targeted to a binding site close to the transcription start site. Using this strategy, and linking peptides selected via phage display, should allow the design of novel DNA-binding proteins--with extraordinary affinity and specificity--for use in biological research and gene therapy.

Zinc finger proteins, of the type first discovered in transcription factor IIIA (TFIIIA), are one of the largest and most important families of DNA-binding proteins. The crystal structure of a complex containing the five Zn fingers from the human GLI oncogene and a high-affinity DNA binding site has been determined at 2.6 $\\angst $ resolution. Finger one does not contact the DNA. Fingers two through five bind in the major groove and wrap around the DNA, but lack the simple, strictly periodic arrangement observed in the Zif268 complex. Fingers four and five of GLI make extensive base contacts in a conserved nine base-pair region, and this section of the DNA has a conformation intermediate between B-DNA and A-DNA. Analyzing the GLI complex and comparing it with Zif268 offers new perspectives on Zn finger-DNA recognition.

The zinc finger DNA-binding motif occurs in many proteins that regulate eukaryotic gene expression. The crystal structure of a complex containing the three zinc fingers from Zif268 (a mouse immediate early protein) and a consensus DNA-binding site has been determined at 2.1 angstroms resolution and refined to a crystallographic R factor of 18.2 percent. In this complex, the zinc fingers bind in the major groove of B-DNA and wrap partway around the double helix. Each finger has a similar relation to the DNA and makes its primary contacts in a three-base pair subsite. Residues from the amino-terminal portion of an α helix contact the bases, and most of the contacts are made with the guanine-rich strand of the DNA. This structure provides a framework for understanding how zinc fingers recognize DNA and suggests that this motif may provide a useful basis for the design of novel DNA-binding proteins.

Key Points C 2 H 2 zinc fingers are the most common DNA-binding motif found in the human genome. The Zif268–DNA crystal structure shows how zinc fingers interact with DNA. The fingers act as modular units (each contacting three to four base pairs of DNA) and the structure reveals which residues should be changed to alter the specificity. Researchers have engineered zinc finger proteins (ZFPs) to bind a diverse set of DNA sequences, and thereby target specific locations in the human genome, such as the promoters of therapeutically relevant genes. Exquisite specificity can be obtained with proteins that have six fingers. ZFP transcription factors (ZFP TFs) are made by combining the ZFPs with domains that either activate or repress genes. ZFP TFs are used in drug discovery to regulate genes for target validation, high-throughput screening and human therapeutics. ZFP-mediated regulation of endogenous genes could make it possible to use genes in a drug discovery application that would otherwise require securing intellectual property rights for a corresponding complementary DNA sequence. Recently, ZFP TFs have been used to promote angiogenesis in a mouse ear model, and are now undergoing further preclinical testing. Combining ZFPs with novel functional domains makes it possible to target DNA for chromatin and DNA modification, DNA cleavage and for targeted integration of exogenous DNA. Zinc-finger proteins (ZFPs) that recognize novel DNA sequences are the basis of a powerful technology platform with many uses in drug discovery and therapeutics. These proteins have been used as the DNA-binding domains of novel transcription factors (ZFP TFs), which are useful for validating genes as drug targets and for engineering cell lines for small-molecule screening and protein production. Recently, they have also been used as a basis for novel human therapeutics. Most of our advances in the design and application of these ZFP TFs rely on our ability to engineer ZFPs that bind short stretches of DNA (typically 9–18 base pairs) located within the promoters of target genes. Here, we summarize the methods used to design these DNA-binding domains, explain how they are incorporated into novel transcription factors (and other useful molecules) and describe some key applications in drug discovery.