Figure 2. Engineering and characterization of ZFNs.

A. Schematic overview of Oligomerized Pool Engineering (OPEN) strategy for constructing zinc finger arrays. Individual zinc fingers are depicted as spheres and DNA target sites as rectangles.

B. Schematic of the human β-globin locus showing the position of the two ZFN target sites in relation to the sickle cell anemia mutation (asterisk) and to the insertion site of the selectable marker (arrow). Exon represented by thick line, introns by thin lines. Not drawn to scale.

C. Recognition helix sequences and bacterial two-hybrid (B2H) fold-activation values of selected zinc finger proteins engineered by OPEN. Amino acid sequence of the recognition helices are shown below their respective three base pair target DNA sites. B2H fold-activation values are shown as blue bars on the right.

D. Cleavage efficiency of ZFN pairs as measured by induction of mutations caused by non-homologous end-joining (NHEJ) repair in HEK293 cells (for site 1) or primary human fibroblasts (for site 2). Wild-type sequence is shown with zinc finger binding sites highlighted in yellow. Mutant sequences are shown below with deletions indicated by dashes and insertions shown in red, lower-case letters. The frequency with which each sequence was detected is indicated to the right.