An improved predictive recognition model for Cys(2)-His(2) zinc finger proteins.

Cys(2)-His(2) zinc finger proteins (ZFPs) are the largest family of transcription factors in higher metazoans. They also represent the most diverse family with regards to the composition of their recognition sequences. Although there are a number of ZFPs with characterized DNA-binding preferences, the specificity of the vast majority of ZFPs is unknown and cannot be directly inferred by homology due to the diversity of recognition residues present within individual fingers.

Using defined finger-finger interfaces as units of assembly for constructing zinc-finger nucleases.

Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger-finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the validation of a new set of two-finger modules that can be used for building ZFAs via conventional assembly methods or a new strategy-finger stitching-that increases the diversity of genomic sequences targetable by ZFNs.

An optimized two-finger archive for ZFN-mediated gene targeting.

The widespread use of zinc-finger nucleases (ZFNs) for genome engineering is hampered by the fact that only a subset of sequences can be efficiently recognized using published finger archives. We describe a set of validated two-finger modules that complement existing finger archives and expand the range of ZFN-accessible sequences threefold. Using this archive, we introduced lesions at 9 of 11 target sites in the zebrafish genome.

Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish.

Zinc-finger nucleases (ZFNs) allow targeted gene inactivation in a wide range of model organisms. However, construction of target-specific ZFNs is technically challenging. Here, we evaluate a straightforward modular assembly-based approach for ZFN construction and gene inactivation in zebrafish.