FIGURE 1

(a) Action of Zinc Finger nucleases (ZFN). ZFN are recombinant proteins that consist of multimers of single Zinc Finger protein (ZFP) shown as ribbon diagrams each containing an helix and zinc ion (blue circle) fused to a restriction enzyme, FokI (red). Each of these Zinc Fingers recognize a specific set of nucleotides (3–4) on either sense or antisense strand of the DNA. Here eight different Zinc Finger proteins with differing specificities are shown. Multimerization of Zinc Fingers confers high specificity of nucleotide sequence recognition. By modular design, two complementary ZFN target FokI to the same region of the sense (light blue) and antisense (dark blue) strand of DNA. This induces ragged cleavage of both strands. Repair mechanisms result in deletion of single to multiple nucleotides from both strands. Non-homologous recombination can also result in insertions (orange and red strands). Both of these mechanisms result in disrupted gene function. (b) Adenovirus delivery of ZFN-targeting CCR5. HIV uses a combination of CD4 and CCR5 to enter the cell. A recombinant adenovirus is used to deliver the coding sequence of the ZFN. Its payload is transferred to the nucleus and transcribed to mRNA coding the ZFN, which is then synthesized in the cytoplasm. The ZFN then enters the nucleus and specifically targets and disrupts the CCR5 gene as in (a). Defective CCR5 protein is degraded in the cytoplasm and is therefore not expressed on the cell membrane so HIV entry fails.