(623t) Engineering Zinc Finger Proteins Using Accessory Binding Modules

We report on the engineering of zinc finger proteins (ZFPs) as synthetic transcription factors by appending accessory modules to enhance protein binding affinity to target DNA. Over the last few years, transcription factor engineering has provided a valuable approach to genetically engineer a wide variety of cell types. ZFP technology is now being used to directly edit chromosomal DNA by creating synthetic nucleases. Recently, this approach has been used to produce genetically enhanced crops and improved biophama products and to cure genetic disease. The central feature of this technology is generation of synthetic transcription factors that bind to "target" DNA sequences with high specificity. In this way, modular assembly provides a convenient approach to synthesize target-specific ZFPs by generating modular proteins consisting of tandem repeats of well-characterized zinc fingers through standard molecular cloning. Oligomerized pool engineering (OPEN) and context-dependent assembly (CoDA) are alternative, albeit more complex protocols for creating ZFPs.

In this work, we describe the direct engineering of two- and three-finger ZFPs to improve DNA binding site recognition specificity and affinity. We generated chimeric ZFPs containing the accessory module PAR from the yeast transcriptional activator ADR1, a zinc finger transcription factor from Saccharomyces cerevisiae. We observed that addition of the PAR binding module increased the binding affinity for chimeric ZFPs to target DNA sequences without affecting the selectivity for two-finger ZFPs. Two-finger ZFPs are generally not used for targeted DNA binding, because the functionality of ZFPs containing only two modules results in a loss in binding affinity and selectivity. Here, we show that ZFPs consisting of only two-finger modules can be successfully engineered to target specific DNA sequences by appending accessory binding modules. Based on this work, we anticipate that these chimeric ZFPs will enable new applications involving genetic modification. When combined with the CoDA protocol for ZFP assembly, pre-screened two-finger ZFPs can be used as simple “unit building blocks”, thereby enabling enhanced design and engineering of target-specific and tight binding synthetic transcription factors.