714. Structure-Based Engineering of the FokI DNA Cleavage Domain in the Context of a Chimeric Endonuclease: Towards Maximally Efficient “Gene Editing” Therapy

Abstract

Nucleases that introduce a double-strand break into a sequence of one's choosing for use in editing (Wilson, Nat. Biotech. 21: 759) can be created by fusing the cleavage domain of the type IIs restriction enzyme FokI to engineered zinc finger proteins (ZFPs). The FokI cleavage domain must dimerize in order to cleave DNA efficiently and therefore two different ZFP-FokI fusions are required to introduce a sequence-specific double-stranded break in a given locus. Creating FokI cleavage domain variants that do not function as homodimers would help ensure that these nucleases do not cleave unintended sites and thus enhance the specificity of the resulting proteins. Using structure-based design, we have created a FokI cleavage domain mutant that can cleave DNA efficiently when paired with a wild-type cleavage domain, but does not function when paired with itself. A second cleavage domain mutant was created that functions when paired with the first mutant, but does not function when paired with itself or with a wild-type cleavage domain. Preliminary experiments using a cell-based gene correction assay indicate that pairs of ZFPs containing these modified FokI cleavage domains robustly potentiate the correction of a chromosomally integrated GFP reporter inactivated with a nonsense mutation. Both proteins were inactive for targeting when used alone in a cellular context, illuminating the utility of a protein engineering approach in the context of a genome editing therapy protocol.