Zinc finger nucleases (ZFNs) have been used to direct precise modifications of the genetic information in living cells at high efficiency. An important consideration in the design of ZFNs is the number of zinc fingers that are required for efficient and specific cleavage. We examined dimeric ZFNs composed of +, +, +, +, +, and + zinc fingers, targeting 6, 12, 18, 24, 30, and 36 bp, respectively. We found that + and + fingers supported neither in vitro cleavage nor single-strand annealing in a cell-based recombination assay. An optimal ZFN activity was observed for + and + fingers. Surprisingly, + and + fingers exhibited significantly reduced activity. While the extra fingers were not found to dramatically increase toxicity, directly inhibit recombination, or perturb the ZFN target site, we demonstrate the ability of subsets of three fingers in six-finger arrays to bind independently to regions of the target site, possibly explaining the decrease in activity. These results have important implications for the design of new ZFNs, as they show that in some cases an excess of fingers may actually negatively affect the performance of engineered multifinger proteins. Maximal ZFN activity will require an optimization of both DNA binding affinity and specificity.
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