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The discovery of zinc fingers and their development for practical applications in gene regulation and genome manipulation

  • Aaron Klug (a1)


A long-standing goal of molecular biologists has been to construct DNA-binding proteins for the control of gene expression. The classical Cys2His2 (C2H2) zinc finger design is ideally suited for such purposes. Discriminating between closely related DNA sequences both in vitro and in vivo, this naturally occurring design was adopted for engineering zinc finger proteins (ZFPs) to target genes specifically.

Zinc fingers were discovered in 1985, arising from the interpretation of our biochemical studies on the interaction of the Xenopus protein transcription factor IIIA (TFIIIA) with 5S RNA. Subsequent structural studies revealed its three-dimensional structure and its interaction with DNA. Each finger constitutes a self-contained domain stabilized by a zinc (Zn) ion ligated to a pair of cysteines and a pair of histidines and also by an inner structural hydrophobic core. This discovery showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA-binding proteins generally make use of the 2-fold symmetry of the double helix, functioning as homo- or heterodimers, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of varying lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that the zinc finger is found widespread in nature, including 3% of the genes of the human genome.

The zinc finger design can be used to construct DNA-binding proteins for specific intervention in gene expression. By fusing selected zinc finger peptides to repression or activation domains, genes can be selectively switched off or on by targeting the peptide to the desired gene target. It was also suggested that by combining an appropriate zinc finger peptide with other effector or functional domains, e.g. from nucleases or integrases to form chimaeric proteins, genomes could be modified or manipulated.

The first example of the power of the method was published in 1994 when a three-finger protein was constructed to block the expression of a human oncogene transformed into a mouse cell line. The same paper also described how a reporter gene was activated by targeting an inserted 9-base pair (bp) sequence, which acts as the promoter. Thus, by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. It was also suggested that, by combining zinc fingers with other effector or functional domains, e.g. from nucleases or integrases, to form chimaeric proteins, genomes could be manipulated or modified.

Several applications of such engineered ZFPs are described here, including some of therapeutic importance, and also their adaptation for breeding improved crop plants.


Corresponding author

*Author for correspondence: A. Klug, MRC Laboratory of Molecular Biology, CambridgeCB2 0QH, UK. Tel.: 01223 252940; Fax: 01223 213556; Email:


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Bateman, A., Birney, E., Cerruti, L., Durbin, R., Etwiller, L., Eddy, S. R., Griffiths-Jones, S., Howe, K. L., Marshall, M. & Sonnhammer, E. L. (2002). The Pfam protein families database. Nucleic Acids Research 30(1), 276280.
Beerli, R. R., Schopfer, U., Dreier, B. & Barbas, C. F. III ( 2000). Chemically regulated zinc finger transcription factors. Journal of Biological Chemistry 275(42), 3261732627.
Berg, J. M. (1988). Proposed structure for the zinc-binding domains from transcription factor IIIA and related proteins. Proceedings of the National Academy of Sciences USA 85(1), 99102.
Bibikova, M., Beumer, K., Trautman, J. K. & Carroll, D. (2003). Enhancing gene targeting with designed zinc finger nucleases. Science 300(5620), 764.
Brown, D. D. (1984). The role of stable complexes that repress and activate eucaryotic genes. Cell 37(2), 359365.
Capecchi, M. R. (1989). Altering the genome by homologous recombination. Science 244(4910), 12881292.
Cavazzana-Calvo, M., Hacein-Bey, S., De Saint Basile, G., Gross, F., Yvon, E., Nusbaum, P., Selz, F., Hue, C., Certain, S., Casanova, J. L., Bousso, P., Deist, F. L. & Fischer, A. (2000). Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288(5466), 669672.
Choo, Y. & Klug, A. (1994a). Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions. Proceedings of the National Academy of Sciences USA 91(23), 1116811172.
Choo, Y. & Klug, A. (1994b). Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage. Proceedings of the National Academy of Sciences USA 91(23), 1116311167.
Choo, Y., Sanchez-Garcia, I. & Klug, A. (1994). In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence. Nature 372(6507), 642645.
Doyon, Y., Mccammon, J. M., Miller, J. C., Faraji, F., Ngo, C., Katibah, G. E., Amora, R., Hocking, T. D., Zhang, L., Rebar, E. J., Gregory, P. D., Urnov, F. D. & Amacher, S. L. (2008). Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nature Biotechnology 26(6), 702708.
Elrod-Erickson, M., Benson, T. E. & Pabo, C. O. (1998). High-resolution structures of variant Zif268-DNA complexes: implications for understanding zinc finger-DNA recognition. Structure 6(4), 451464.
Fairall, L., Schwabe, J. W., Chapman, L., Finch, J. T. & Rhodes, D. (1993). The crystal structure of a two zinc-finger peptide reveals an extension to the rules for zinc-finger/DNA recognition. Nature 366(6454), 483487.
Ginsberg, A. M., King, B. O. & Roeder, R. G. (1984). Xenopus 5S gene transcription factor, TFIIIA: characterization of a cDNA clone and measurement of RNA levels throughout development. Cell 39(3 Pt 2), 479489.
Hanas, J. S., Hazuda, D. J., Bogenhagen, D. F., Wu, F. Y. & Wu, C. W. (1983). Xenopus transcription factor A requires zinc for binding to the 5 S RNA gene. Journal of Biological Chemistry 258(23), 1412014125.
Isalan, M., Choo, Y. & Klug, A. (1997). Synergy between adjacent zinc fingers in sequence-specific DNA recognition. Proceedings of the National Academy of Sciences USA 94(11), 56175621.
Isalan, M., Klug, A. & Choo, Y. (1998). Comprehensive DNA recognition through concerted interactions from adjacent zinc fingers. Biochemistry 37(35), 1202612033.
Isalan, M., Klug, A. & Choo, Y. (2001). A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nature Biotechnology 19(7), 656660.
Jamieson, A. C., Miller, J. C. & Pabo, C. O. (2003). Drug discovery with engineered zinc-finger proteins. Nature Reviews Drug Discovery 2(5), 361368.
Jasin, M. (1996). Genetic manipulation of genomes with rare-cutting endonucleases. Trends in Genetics 12(6), 224228.
Kim, J. S. & Pabo, C. O. (1998). Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants. Proceedings of the National Academy of Sciences USA 95(6), 28122817.
Kim, Y. G., Cha, J. & Chandrasegaran, S. (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proceedings of the National Academy of Sciences USA 93(3), 11561160.
Klug, A. (1983). From macromolecules to biological assemblies. In Les Prix Nobel en 1982, pp. 93125. Stockholm: Nobel Foundation.
Klug, A., Jack, A., Viswamitra, M. A., Kennard, O., Shakked, Z. & Steitz, T. A. (1979). A hypothesis on a specific sequence-dependent conformation of DNA and its relation to the binding of the lac-repressor protein. Journal of Molecular Biology 131(4), 669680.
Kornberg, R. D. (1977). Structure of chromatin. Annual Review of Biochemistry 46, 931954.
Lee, M. S., Gippert, G. P., Soman, K. V., Case, D. A. & Wright, P. E. (1989). 3-Dimensional solution structure of a single zinc finger DNA-binding domain. Science 245(4918), 635637.
Liu, P. Q., Rebar, E. J., Zhang, L., Liu, Q., Jamieson, A. C., Liang, Y., Qi, H., Li, P. X., Chen, B., Mendel, M. C., Zhong, X., Lee, Y. L., Eisenberg, S. P., Spratt, S. K., Case, C. C. & Wolffe, A. P. (2001). Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A. Journal of Biological Chemistry 276(14), 1132311334.
McCafferty, J., Griffiths, A. D., Winter, G. & Chiswell, D. J. (1990). Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348(6301), 552554.
Meng, X., Noyes, M. B., Zhu, L. J., Lawson, N. D. & Wolfe, S. A. (2008). Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nature Biotechnology 26(6), 695701.
Miller, J., Mclachlan, A. D. & Klug, A. (1985). Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO Journal 4(6), 16091614.
Moore, M., Klug, A. & Choo, Y. (2001). Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units. Proceedings of the National Academy of Sciences USA 98(4), 14371441.
Neuhaus, D., Nakaseko, Y., Schwabe, J. W. R. & Klug, A. (1992). Solution structures of 2 zinc-finger domains from Swi5 obtained using 2-dimensional H-1 nuclear-magnetic-resonance spectroscopy – a zinc-finger structure with a 3rd strand of beta-sheet. Journal of Molecular Biology 228(2), 637651.
Papworth, M., Moore, M., Isalan, M., Minczuk, M., Choo, Y. & Klug, A. (2003). Inhibition of herpes simplex virus 1 gene expression by designer zinc-finger transcription factors. Proceedings of the National Academy of Sciences USA 100(4), 16211626.
Pavletich, N. P. & Pabo, C. O. (1991). Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2·1 A. Science 252(5007), 809817.
Pelham, H. R. & Brown, D. D. (1980). A specific transcription factor that can bind either the 5S RNA gene or 5S RNA. Proceedings of the National Academy of Sciences USA 77(7), 41704174.
Perez, E. E., Wang, J., Miller, J. C., Jouvenot, Y., Kim, K. A., Liu, O., Wang, N., Lee, G., Bartsevich, V. V., Lee, Y. L., Guschin, D. Y., Rupniewski, I., Waite, A. J., Carpenito, C., Carroll, R. G., Orange, J. S., Urnov, F. D., Rebar, E. J., Ando, D., Gregory, P. D., Riley, J. L., Holmes, M. C. & June, C. H. (2008). Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nature Biotechnology 26(7), 808816.
Picard, B. & Wegnez, M. (1979). Isolation of a 7S particle from Xenopus laevis oocytes: a 5S RNA-protein complex. Proceedings of the National Academy of Sciences USA 76(1), 241245.
Porteus, M. H. & Baltimore, D. (2003). Chimeric nucleases stimulate gene targeting in human cells. Science 300(5620), 763.
Rebar, E. J., Huang, Y., Hickey, R., Nath, A. K., Meoli, D., Nath, S., Chen, B., Xu, L., Liang, Y., Jamieson, A. C., Zhang, L., Spratt, S. K., Case, C. C., Wolffe, A. & Giordano, F. J. (2002). Induction of angiogenesis in a mouse model using engineered transcription factors. Nature Medicine 8(12), 14271432.
Reynolds, L., Ullman, C., Moore, M., Isalan, M., West, M. J., Clapham, P., Klug, A. & Choo, Y. (2003). Repression of the HIV-1 5′ LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors. Proceedings of the National Academy of Sciences USA 100(4), 16151620.
Rhodes, D. & Klug, A. (1981). Sequence-dependent helical periodicity of DNA. Nature 292(5821), 378380.
Rosenberg, U. B., Schroder, C., Preiss, A., Kienlin, A., Cote, S., Riede, I. & Jackle, H. (1986). Structural homology of the product of the Drosophila Kruppel gene with Xenopus transcription factor-Iiia. Nature 319(6051), 336339.
Santiago, Y., Chan, E., Liu, P. Q., Orlando, S., Zhang, L., Urnov, F. D., Holmes, M. C., Guschin, D., Waite, A., Miller, J. C., Rebar, E. J., Gregory, P. D., Klug, A. & Collingwood, T. N. (2008). Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proceedings of the National Academy of Sciences USA 105(15), 58095814.
Shukla, V. K., Doyon, Y., Miller, J. C., Dekelver, R. C., Moehle, E. A., Worden, S. E., Mitchell, J. C., Arnold, N. L., Gopalan, S., Meng, X., Choi, V. M., Rock, J. M., Wu, Y. Y., Katibah, G. E., Zhifang, G., Mccaskill, D., Simpson, M. A., Blakeslee, B., Greenwalt, S. A., Butler, H. J., Hinkley, S. J., Zhang, L., Rebar, E. J., Gregory, P. D. & Urnov, F. D. (2009). Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459(7245), 437441.
Smith, D. R., Jackson, I. J. & Brown, D. D. (1984). Domains of the positive transcription factor specific for the Xenopus 5S RNA gene. Cell 37(2), 645652.
Smith, G. P. (1985). Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228(4705), 13151317.
Tan, S., Guschin, D., Davalos, A., Lee, Y. L., Snowden, A. W., Jouvenot, Y., Zhang, H. S., Howes, K., Mcnamara, A. R., Lai, A., Ullman, C., Reynolds, L., Moore, M., Isalan, M., Berg, L. P., Campos, B., Qi, H., Spratt, S. K., Case, C. C., Pabo, C. O., Campisi, J. & Gregory, P. D. (2003). Zinc-finger protein-targeted gene regulation: genomewide single-gene specificity. Proceedings of the National Academy of Sciences USA 100(21), 1199712002.
Townsend, J. A., Wright, D. A., Winfrey, R. J., Fu, F., Maeder, M. L., Joung, J. K. & Voytas, D. F. (2009). High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459(7245), 442445.
Tso, J. Y., Vandenberg, D. J. & Korn, L. J. (1986). Structure of the Gene for Xenopus Transcription Factor Tfiiia. Nucleic Acids Research 14(5), 21872200.
Urnov, F. D., Miller, J. C., Lee, Y. L., Beausejour, C. M., Rock, J. M., Augustus, S., Jamieson, A. C., Porteus, M. H., Gregory, P. D. & Holmes, M. C. (2005). Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435(7042), 646651.
Vincent, A., Colot, H. V. & Rosbash, M. (1985). Sequence and structure of the serendipity locus of Drosophila melanogaster. A densely transcribed region including a blastoderm-specific gene. Journal of Molecular Biology 186(1), 149166.

The discovery of zinc fingers and their development for practical applications in gene regulation and genome manipulation

  • Aaron Klug (a1)


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