Evaluation of novel design strategies for developing zinc finger nucleases tools for treating human diseases.

Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable tools to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger.

Strategy for naturelike designer transcription factors with reduced toxicity.

For clinical applications, the biological functions of DNA-binding proteins require that they interact with their target binding site with high affinity and specificity. Advances in randomized production and target-oriented selection of engineered artificial DNA-binding domains incited a rapidly expanding field of designer transcription factors (TFs). Engineered transcription factors are used in zinc-finger nuclease (ZFN) technology that allows targeted genome editing. Zinc-finger-binding domains fabricated by modular assembly display an unexpectedly high failure rate having either a lack of activity as ZFNs in human cells or activity at "off-target” binding sites on the human genome causing cell death. To address these shortcomings, we created new binding domains using a targeted modification strategy. We produced two SP1 mutants by exchanging amino acid residues in the alpha-helical region of the transcription factor SP1. We identified their best target binding sites and searched the NCBI HuRef genome for matches of the nine-base-pair consensus binding site of SP1 and the best binding sites of its mutants. Our research concludes that we can alter the binding preference of existing zinc-finger domains without altering its biological functionalities.