ZiFDB version 1.0

Department of Genetics, Development and Cell Biology

Introduction

The C₂H₂ zinc finger motif, which was discovered in transcription factor TFIIIA from Xenopus laevis (Miller et al., 1985), is one of the most abundant DNA-binding motifs in nature. Each zinc finger comprises about 30 amino acids that fold into a ßßα structure through hydrophobic interactions and binding of a zinc ion by two conserved cysteine and histidine residues. Each zinc finger motif typically recognizes a continuous 3_bp DNA sequence. The modularity of its structure and DNA binding activity make the zinc finger motif a good scaffold for constructing engineered DNA binding proteins.

The methods used to engineer zinc finger arrays (ZFAs) that recognize novel target sequences can be classified into two categories: selection methods and modular assembly. Modular assembly is fast and easy: it simply involves linking together zinc finger modules that rocognize known target sites. At present, however, modular assembly is not adequately reliable. For example, more than half of the ZFAs created by modular assembly have little or no activity (Ramirez et al., 2008). We hypothesize that there are yet undefined rules, which once understood, will make the engineering of ZFAs by modular assembly more reliable.

In contrast to modular assembly, selection methods are much more reliable, however, they often require considerable time and a high level of molecular biology expertise. The Joung lab at Harvard Medical School, recently implemented a seletion-based platform called OPEN (Oligomerizd Pool Engineering) that is considerably easier than previously-described strategies (Maeder et al. 2008). We anticipate that large numbers of ZFAs will be generated by OPEN in the coming years.

Of course the simplest way to obtain a ZFA for a target site of choice is to use an array that has been previously characterized by another group. The establishment of ZiFDB was partially motivated by a desire to aid molecular biologists in determining if arrays (or partial arrays) exist for target sites under consideration. Another motivation for ZiFDB was the belief that if all zinc fingers and ZFAs are organized in a central archive, it may be possible to establish better rules for engineering by modular assembly and for understanding how ZFAs recognize their DNA targets.

ZiFDB organizes information on both individual zinc finger modules and engineered ZFAs. There are currently four sets of zinc finger modules available: 1) Sangamo BioScience researchers have identified fingers recognizing all 5'-GNN-3' and a few of 5'-ANN-3', 5'-CNN-3' and 5'-TNN-3' triplets using phage display, targeted mutagenesis and SELECT methods (Liu et al., 2002); 2) the Barbas group constructed another set of models, which recognize all 5'-GNN-3', most 5'-ANN-3', 5'-CNN-3' and a few 5'-TNN-3' triplets (Dreier et al., 2001; Dreier et al., 2005; Dreier et al., 2000;); 3) Toolgen, Inc. isolated a set of naturally-occurring zinc finger modules from human transcription factors (Bae et al., 2003); 4) the Joung lab has made a large number of ZFAs by OPEN, and the constituent zinc fingers are included in the database. For the engineered ZFAs, we have collected information on 3-finger ZFAs, since this is the architecture advocated by the Zinc Finger Consortium (http://www.zincfingers.org), a group of academic laboratories dedicated to the development of improved methods to engineer zinc finger proteins. Currently, all ZFAs in ZiFDB are described in the published literature. In the future, unpublished ZFAs will also be included. It is hoped that the information in this database will help molecular biologists develop zinc finger reagents that meet their needs for genome modification. Further, we hope the analysis of the collected information will aid in improving modular design.

References

Ramirez, C.L., Foley, J.E., Wright, D.A., Müller-Lerch, F., Rahman, S.H., Cornu, T.I., Winfrey, R.J., Sander, J.D., Fu, F., Townsend, J.A., Cathomen, T., Voytas, D.F., Joung, J.K. (2008) Unexpected failure rates for modular assembly of engineered zinc-fngers. Nature Methods, 5:374

Dreier, B., Fuller, R.P., Segal, D.J., Lund, C.V., Blancafort, P., Huber, A., Koksch, B. and Barbas, C.F., 3rd. (2005) Development of zinc finger domains for recognition of the 5'-CNN-3' family DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem, 280, 35588-35597.

Bae, K.H., Kwon, Y.D., Shin, H.C., Hwang, M.S., Ryu, E.H., Park, K.S., Yang, H.Y., Lee, D.K., Lee, Y., Park, J. et al. (2003) Human zinc fingers as building blocks in the construction of artificial transcription factors. Nat Biotechnol, 21, 275-280.

Liu, Q., Xia, Z., Zhong, X. and Case, C.C. (2002) Validated zinc finger protein designs for all 16 GNN DNA triplet targets. J Biol Chem, 277, 3850-3856.

Dreier, B., Beerli, R.R., Segal, D.J., Flippin, J.D. and Barbas, C.F., 3rd. (2001) Development of zinc finger domains for recognition of the 5'-ANN-3' family of DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem, 276, 29466-29478.

Maeder, M.L., Thibodeau-Beganny, S., Osiak, A., Wright, D.A., Anthony, R.M., Eichtinger, M., Jiang, T., Foley, J.E., Winfrey, R.J., Townsend, J.A., et al. (2008). Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Molecular cell 31, 294-301.

Miller, J., McLachlan, A. D., and Klug, A. (1985). Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. Embo J 4, 1609-1614.

If you use ZiFDB please reference:Zinc Finger Database (ZiFDB): a repository for information on C2H2 zinc fingers and engineered zinc- finger arrays. Fengli Fu; Jeffry D. Sander; Morgan Maeder; Stacey Thibodeau-Beganny; J. Keith Joung; Drena Dobbs; Leslie Miller; Daniel F. Voytas. Nucleic Acids Research 2008; doi: 10.1093/nar/gkn606

This material is based upon work supported by National Science Foundation Grant No. DBI0501678. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation (NSF).

Voytas Laboratory
University of Minnesota
Phone:(612) 626-4509
Email: voytas@umn.edu