C2H2-zinc fingers (ZFs) are commonly found in transcription factors that code for nearly 3% of gene products in the human genome. ZF proteins are commonly involved in gene regulation during development, cell differentiation, and tumor suppression. Each "finger" is a domain composed of approximately 30 amino acids. Since the discovery of these domains over 25 years ago, several groups have contributed to the structural and biochemical knowledge to understand their DNA-binding properties. Taking advantage of the simplicity of manipulating the DNA-binding potential of a ZF, the technology has now evolved to make sequence-specific Zinc Finger Nucleases (ZFNs), Artificial Transcription Factors (ATFs), Zinc Finger Recombinases, and DNA detection tools. ZFPs have been used for various applications, ranging from regulating genes by ZF-ATFs to manipulating genomes in diverse organisms. ZFNs have remarkably revolutionized the field of genome engineering. ZFN-modified T-cells have now advanced into human clinical trials for cell-based therapies as a treatment against HIV. Despite the advances in the ZFN technology, one of the challenges in the field is obtaining effective ZFNs using publicly available tools. The traditional method of synthesizing custom ZF arrays was using modular assembly (MA). In this method, preselected ZFs from publicly available one-finger archives can be assembled modularly to make long arrays. MA of ZFNs provides a rapid method to create proteins that can recognize a broad spectrum of DNA sequences. However, three- and four-finger arrays often fail to produce active nucleases. The low success rate of MA ZF arrays was attributed to the fact that they suffer from finger-finger incompatibility referred to as context-dependent effects. However, we hypothesized that the low affinity of MA arrays was the limiting factor. The work presented in this dissertation describes our efforts at addressing these fundamental methodological challenges. We developed the Extended Modular Assembly method that overcomes the limitations of both the previous Modular Assembly. We performed a systematic investigation of number and composition of modules on ZFN activity and analyzed ZFN specificity both in vitro and in vivo. Our current experiments apply the ZFNs produced by our method to study the role of genetic variation in human disease.

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