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Genome engineering through homologous recombination (HR) is a powerful instrument for studying biological pathways or creating treatment options for genetic disorders. In mammalian cells HR is rare but the creation of targeted DNA double-strand breaks stimulates HR significantly. Here, we present a method to generate, evaluate, and optimize rationally designed endonucleases that promote HR. The DNA-binding domains were synthesized by assembling predefined zinc-finger modules selected by phage display. Attachment of a transcriptional activation domain allowed assessment of DNA binding in reporter assays, while fusion with an endonuclease domain created custom nucleases that were tested for their ability to stimulate HR in episomal and chromosomal gene repair assays. We demonstrate that specificity, expression kinetics, and protein design are crucial parameters for efficient gene repair and that our two-step assay allows one to go quickly from design to testing to successful employment of the custom nucleases in human cells.

N-Acetylneuraminic acid (Neu5Ac) is the precursor of sialic acids, a group of important molecules in biological recognition systems. Biosynthesis of Neu5Ac is initiated and regulated by its key enzyme, UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC 5.1. 3.14)/N-acetylmannosamine kinase (ManNAc kinase, EC 2.7.1.60) in rat liver (Hinderlich, S., Stasche, R., Zeitler, R., and Reutter, W. (1997) J. Biol. Chem. 272, 24313-24318). In the present paper we report the isolation and characterization of a cDNA clone encoding this bifunctional enzyme. An open reading frame of 2166 base pairs encodes 722 amino acids with a predicted molecular mass of 79 kDa. The deduced amino acid sequence contains exact matches of the sequences of five peptides derived from tryptic cleavage of the enzyme. The recombinant bifunctional enzyme was expressed in COS7 cells, where it displayed both epimerase and kinase activity. Distribution of UDP-GlcNAc 2-epimerase/ManNAc kinase in the cytosol of several rat tissues was investigated by determining both specific enzyme activities. Secreting organs (liver, salivary glands, and intestinal mucosa) showed high specific activities of UDP-GlcNAc 2-epimerase/ManNAc kinase, whereas significant levels of these activities were absent from other organs (lung, kidney, spleen, brain, heart, skeletal muscle, and testis). Northern blot analysis revealed no UDP-GlcNAc 2-epimerase/ManNAc kinase mRNA in the non-secreting tissues.

In previous studies, we have developed a technology for the rapid construction of novel DNA-binding proteins with the potential to recognize any unique site in a given genome. This technology relies on the modular assembly of modified zinc finger DNA-binding domains, each of which recognizes a three bp subsite of DNA. A complete set of 64 domains would provide comprehensive recognition of any desired DNA sequence, and new proteins could be assembled by any laboratory in a matter of hours. However, a critical parameter for this approach is the extent to which each domain functions as an independent, modular unit, without influence or dependence on its neighboring domains. We therefore examined the detailed binding behavior of several modularly assembled polydactyl zinc finger proteins. We first demonstrated that 80 modularly assembled 3-finger proteins can recognize their DNA target with very high specificity using a multitarget ELISA-based specificity assay. A more detailed analysis of DNA binding specificity for eight 3-finger proteins and two 6-finger proteins was performed using a target site selection assay. Results showed that the specificity of these proteins was as good or better than that of zinc finger proteins constructed using methods that allow for interdependency. In some cases, near perfect specificity was achieved. Complications due to target site overlap were found to be restricted to only one particular amino acid interaction (involving an aspartate in position 2 of the alpha-helix) that occurs in a minority of cases. As this is the first report of target site selection for designed, well characterized 6-finger proteins, unique insights are discussed concerning the relationship of protein length and specificity. These results have important implications for the design of proteins that can recognize extended DNA sequences, as well as provide insights into the general rules of recognition for naturally occurring zinc finger proteins.

Genome engineering is a powerful instrument to study basic biological questions or to create treatment options for genetic diseases. A safe way to correct genetic mutations is through homologous recombination (HR), during which a mutant sequence is replaced with the corresponding wild-type sequence. While HR is predominant in yeast, it is a rare event in mammalian cells where non-homologous recombination is much more frequent. The creation of a targeted DNA double-strand break (DSB) has been shown to locally stimulate HR several hundred-fold by activating the cellular machinery for double-strand break repair, but it presupposes the availability of a site-specific endonuclease to create the DSB. Some initial experiments with artificial nucleases in human cells revealed that cytotoxicity of such enzymes is a major issue, most likely through cleavage at non-specific sites. Here, we present a rapid two-step method to evaluate and optimize novel, rationally designed endonucleases. In a first step, the DNA-binding domain was synthesized by assembling predefined C2H2-zinc-finger modules and fused to a transcriptional activation domain. Each zinc-finger module is designed to recognize 3 bp of DNA and the modules can be assembled in any order necessary to recognize any given sequence in a target locus. DNA-binding specificity of these artificial transcription factors was assessed in a reporter assay. In a second step, the DNA-binding domain was attached to the catalytic domain of the FokI restriction endonuclease to generate the custom nucleases. Upon co-transfection with a repair matrix, they were assessed for their ability to stimulate HR in an episomal gene repair assay. We have engineered several kinds of custom nucleases and demonstrate that high specificity, fine-tuned expression kinetics and protein design are absolutely critical to promote HR. Importantly, the same parameters are also crucial for a low level of cytotoxicity. After optimization, expression of the custom nucleases stimulated both episomal and chromosomal gene repair and HR was achieved in 35% or 2.5% of transfected cells, respectively. Thus, custom nucleases will be a valuable tool to stimulate gene repair for therapeutic purposes. Moreover, they can be easily combined with viral vector systems for efficient delivery.