(A) OPEN zinc-finger pool construction. Zinc-finger domains are shown as spheres and associated 3 bp subsites as rectangles. Randomized finger in the library is rainbow colored. Note that the figure illustrates how finger pools for the middle position in a three-finger domain were made, but that pools for amino- or carboxy-terminal fingers were also obtained by building libraries in which finger 1 or finger 3 were randomized, respectively (Experimental Procedures).
(B) GXX and TXX target subsites for which finger pools have been constructed (highlighted in grey).
(C) Schematic overview of OPEN selection for a target DNA site. Zinc-fingers and associated subsites represented as in (A). Details in Supplemental Experimental Procedures.
(D) Schematic of the bacterial two-hybrid (B2H) system. ZFP = zinc-finger protein. X and Y = arbitrary interacting proteins.

(A) Quantitative B2H assay of modular assembly (MA; red bars) and OPEN (green bars) zinc finger arrays. Mean fold-activation values (colored bars) and standard deviations (error bars) from three independent assays are shown.
(B) EGFP-disruption assay for testing ZFN activities in human cells.
(C) Modularly assembled and OPEN ZFNs assessed using the EGFP-disruption assay. Error bars represent standard deviations. Single and double asterisks indicate p values <0.05 or <0.01, respectively.

(A) Schematic of CEL I assay for assaying ZFN-induced mutations.
(B-C) Mutation of the endogenous human VEGF-A gene (B) and HoxB13 gene (C) by OPEN ZFNs. Colored arrows indicate expected CEL I digestion products. Images shown are from representative experiments.

(A) OPEN VEGF-A ZFNs and previously described IL2RγZFNs induce efficient gene targeting at endogenous genes in human K562 cells. Top part shows representative gel images from limited-cycle PCR/restriction digest assays and bottom part shows gene targeting frequency means (colored bars) and standard errors (error bars) from multiple experiments.
(B) Gene targeting efficiencies of OPEN VEGF-A ZFNs assessed nine days post-transfection by limited-cycle PCR/restriction digest and Southern blot assays.
(C) Vinblastine enhances gene targeting by OPEN VEGF-A and four-finger IL2RγZFNs. Assays performed four days post-transfection. Data presented as in (A).
(D) Toxicities of OPEN VEGF-A and four-finger IL2RγZFNs in human K562 cells. Means of GFP (green bars) and gene targeting ratios (purple bars) are shown. Error bars represent standard deviations. Single and double asterisks indicate p values <0.05 or <0.01, respectively.
(E) Gene targeting efficiencies of OPEN VEGF-A and four-finger IL2RγZFNs in toxicity experiments of (D). Means and standard deviations (error bars) are shown of PCR-based assays performed four days post-transfection.