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.

miR-221 is required for endothelial tip cell behaviors during vascular development.

Angiogenesis requires coordination of distinct cell behaviors between tip and stalk cells. Although this process is governed by regulatory interactions between the vascular endothelial growth factor (Vegf) and Notch signaling pathways, little is known about the potential role of microRNAs. Through deep sequencing and functional screening in zebrafish, we find that miR-221 is essential for angiogenesis.

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.

Identification of cis regulatory features in the embryonic zebrafish genome through large-scale profiling of H3K4me1 and H3K4me3 binding sites.

An organism's genome sequence serves as a blueprint for the proteins and regulatory RNAs essential for cellular function. The genome also harbors cis-acting non-coding sequences that control gene expression and are essential to coordinate regulatory programs during embryonic development. However, the genome sequence is largely identical between cell types within a multi-cellular organism indicating that factors such as DNA accessibility and chromatin structure play a crucial role in governing cell-specific gene expression.

The human oral microbiome.

The human oral cavity contains a number of different habitats, including the teeth, gingival sulcus, tongue, cheeks, hard and soft palates, and tonsils, which are colonized by bacteria. The oral microbiome is comprised of over 600 prevalent taxa at the species level, with distinct subsets predominating at different habitats. The oral microbiome has been extensively characterized by cultivation and culture-independent molecular methods such as 16S rRNA cloning.

The Human Oral Microbiome Database: a web accessible resource for investigating oral microbe taxonomic and genomic information.

The human oral microbiome is the most studied human microflora, but 53% of the species have not yet been validly named and 35% remain uncultivated. The uncultivated taxa are known primarily from 16S rRNA sequence information. Sequence information tied solely to obscure isolate or clone numbers, and usually lacking accurate phylogenetic placement, is a major impediment to working with human oral microbiome data.