Summary 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 multi-finger 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 to 50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, 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.

An efficient method for making directed DNA sequence modifications to plant genes (gene targeting) is presently lacking, thereby frustrating efforts to dissect plant gene function and engineer crop plants that better produce the world's burgeoning need for food, fiber and fuel. Zinc finger nucleases (ZFNs) - enzymes engineered to create DNA double strand breaks at specific loci - are potent stimulators of gene targeting,1,2 including at engineered reporter genes in plants.3,4 Here we demonstrate high frequency ZFN-stimulated gene targeting at endogenous plant genes, namely the tobacco acetohydroxyacid synthase (SuRA and SuRB) genes, for which specific mutations are known to confer resistance to imidazolinone and sulfonylurea herbicides.5 Herbicide-resistance mutations were introduced into SuR loci by ZFN-mediated gene targeting at frequencies exceeding 2% of transformed cells for mutations as far as 1.3 kb from the ZFN cleavage site. More than 40% of recombinant plants had modifications in multiple SuR alleles. The observed high frequency of gene targeting indicates that it is now possible to efficiently make targeted sequence changes in endogenous plant genes.

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SummaryHomologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases.

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.

In the version of this correspondence initially published, the two previously published datasets analyzed were labeled with incorrect references in Figure 1b. Reference 2 should be associated with the second column (80 sites), and reference 3 should be associated with the third column (96 sites).