Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification

Abstract

Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an "open-source" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.

Figure 1. OPEN Method for Engineering Zinc-finger Arrays

(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.

Figure 2. OPEN ZFNs Engineered to Cleave EGFP Gene Sequences

(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.

Figure 3. Highly Efficient Mutagenesis of Endogenous Human Genes by OPEN ZFNs

(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.

Figure 4. Highly Efficient Gene Targeting of Endogenous Human Loci by OPEN ZFNs

(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.