Gene therapy is a powerful approach for the treatment of disease, however, current therapies using viral vectors carry significant risk of tumorigenesis due to their use of non-specific gene insertion. To meet this challenge, we engineered zinc finger proteins (ZFPs), which are tailored to bind to DNA with high specificity, enabling precise genome editing. Our group has developed a foundational technology for synthesizing nearly 55,000 unique ZFPs using chip-based DNA synthesis based on bioinformatic analysis and for identifying the best binder using a novel genomically encoded 1-hybrid genetic selection scheme in a massively multiplexed fashion. Further, we employed multiplex automated genome engineering (MAGE) for facile editing of the E. coli genome enabling rapid modification of ZFP target sites, gene knockouts and silent codon substitutions. These tools allow for low-cost creation of ZFPs targeting any endogenous human gene, which will increase the accessibility of customized genome editing for gene therapy. While many different natural zinc finger proteins can be employed for synthetic biology-based tasks, engineered zinc finger proteins are most commonly composed of an array of three zinc fingers based on Zif268. Zif268 is a well-characterized mammalian early response transcription factor also known as EGR-1 that was discovered in mice. Zif268 has three C2H2 finger subunits, and each finger binds a specific 3-nucleotide base pair (bp) triplet on a DNA strand. Composed of an array of three fingers, Zif268 binds to three consecutive DNA triplets that in total comprise a 9-bp binding site. This 3-finger domain, 9-bp specificity is a commonly occurring theme in zinc finger literature, although it is by no means a rule. Natural and engineered zinc finger arrays may contain fewer or more zinc fingers and bind DNA sequences of corresponding length and specificity. While any given zinc finger might bind a certain 3-bp DNA sequence with exquisite specificity, even the most specific 3-bp binding event is of little practical value for gene therapy, as a given 3-bp sequence may appear countless times within a given genome. Thus, for practical use in drug delivery or gene therapy, zinc fingers must be engineered to bind more specifically to DNA sequences. Such increased specificity can be achieved through the creation of multi-finger arrays that bind to specific 9-bp sequences. With this in mind, research has focused on creating modular zinc finger subunits, which can be combined into multi-finger arrays. Three-finger arrays, for instance, are tailored to bind to a specific 9-bp sequence through the selection of appropriate zinc fingers to bind to each DNA triplet. Different studies have suggested different models of binding and varying levels of success for zinc finger modularity. Thus, through an approach utilizing data-mining, binding models, and comprehensive bioinformatics, in conjunction with next-generation oligo synthesis technology, we hope to develop a method for engineering zinc finger proteins that can bind to any arbitrarily-selected DNA sequence. The success of such an engineering method would, in turn, facilitate clinical advancements by promoting highly-specific, targeted gene therapies, and would promote personalized medicine by allowing the production of zinc finger-based therapies tailored to unique genomic sequences. Source.