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.
Bibliographical noteFunding Information:
We thank Chong Jin Park for help with statistical calculations and Andrew Hirsh for helpful discussions and comments. J.K.J. is supported by the NIH (R01GM069906, R24GM078369, and R21RR024189), the Cystic Fibrosis Foundation (CFF) (MCCRAY07G0), and the MGH Pathology Service. D.F.V. is supported by the NSF (DBI 0501678). T.C. is supported by the European Commission's 6th Framework Programme (037783 ZNIP). P.B.M. is supported by the CFF (MCCRAY07G0) and the Roy J. Carver Charitable Trust. A.J.I. is supported by the MGH Pathology Service. D.C.S. is supported by the NIH (R01CA112021 and NCI SPORE in Breast Cancer at MGH). M.H.P. is supported by the NIH (R01 HL079295). J.K.J. dedicates this paper to the memory of Robert L. Burghoff and his belief in the power of methods.