An optimized two-finger archive for ZFN-mediated gene targeting - PubMed (original) (raw)

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

Ankit Gupta et al. Nat Methods. 2012 Jun.

Abstract

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.

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Figures

Figure 1

Selection and characterization of 2F-modules recognizing GANNCG sequences. (a) Schematic representation of constructs encoded by the two-finger-ZFP library. Two orthogonal 2F-libraries were constructed each containing randomized amino acids at the finger-finger interface: at positions 5 and 6 of finger 1 (F1) and positions −1, 1 and 2 of finger 2 (F2). The residue at position 3 of F2 was either Asparagine (Asn+3F2-library) or Histidine (His+3F2-library). The arrows indicate canonical interactions between critical specificity determinants on the recognition helix and bases within each finger subsite (b) Schematic representation of bacterial one-hybrid (B1H) based selections to identify 2F-modules specific for a target site. The 2F-libraries were fused between the DNA-binding domain of the Engrailed homeodomain and the ωsubunit of the RNA polymerase. The 6 bp zinc finger target site is present on a His3/Ura3 reporter plasmid between the homeodomain binding site and the −35 box. (c) Montage of DNA-binding specificities for the most favorable 2F-modules with specificity for each GAN-NYG sequence where N-N represent the sixteen possible 2 bp junctions. The 2F-modules are designated as having ‘preferential specificity’ (black dinucleotide) or ‘compatible specificity’ (blue dinucleotide) for the desired target sequence. Further details on these clones are found in Supplementary Fig. 5

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An optimized two-finger archive for ZFN-mediated gene targeting - PubMed (original) (raw)