Engineered zinc-finger nucleases (ZFNs) form the basis of a broadly applicable method for targeted, efficient modification of eukaryotic genomes. In recent work, we described OPEN (oligomerized pool engineering), an 'open-source,' combinatorial selectionbased method for engineering zinc-finger arrays that function well as ZFNs. We have also shown in direct comparisons that the OPEN method has a higher success rate than previously described 'modular-assembly' methods for engineering ZFNs. OPEN selections are carried out in Escherichia coli using a bacterial two-hybrid system and do not require specialized equipment. Here we provide a detailed protocol for carrying out OPEN to engineer zinc-finger arrays that have a high probability of functioning as ZFNs. Using OPEN, researchers can generate multiple, customized ZFNs in ∼8 weeks.
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ACKNOWLEDGMENTS We thank M. Eichtinger, T. Jiang and S. Pruett-Miller for technical assistance and M. Porteus for helpful discussions during early stages of protocol development. M.L.M., S.T.-B. and J.K.J. are supported by grants from the National Institutes of Health (R01 GM069906, R24 GM078369, R21 RR024189) and the MGH Pathology Service. D.F.V. is supported by the National Science Foundation (DBI 0501678). J.D.S. is supported by a Pioneer-Hi-Bred International 2008 Graduate Fellowship, the National Institutes of Health (R33 GM066387) and the Iowa State University CIAG. Note added in proof Shukla et al.78 recently demonstrated ZFN-enhanced gene targeting of an endogenous plant gene.