More than 15 years ago, Srinivasan Chandrasegaran and colleagues found that the Type IIS restriction endonuclease FokI is a two-domain protein, with separable DNA-recognition and cleavage functions. Chandra then began linking the FokI cleavage domain to novel DNA-binding domains and showing altered specificity of cleavage. When he used a binding domain comprised of zinc fingers, we became very interested in these hybrids because we knew that ZFs are very flexible in their recognition of DNA.
The Cys2His2 zinc finger is a module of about 28-30 amino acids that is found in a large family of eukaryotic transcription factors. Its structure consists of an a-helix, two b-sheets, and a single zinc atom. Each finger recognizes principally three base pairs of DNA through contacts in the major groove. The recognition elements in the protein are side chains of amino acids at three positions, specified as -1, 2 and 6 relative to the N-terminus of the a-helix. Changing the identities of these residues (with some influence from surrounding ones) alters the DNA-recognition specificity of the finger. Workers in a number of labs have identified some natural and many derived fingers that recognize many of the 64 triplets. To a large extent, each ZF binds DNA independently of the surrounding fingers, so existing fingers can be rearranged to recognize new sequences. It takes at least 3 fingers to provide enough contacts to bind DNA with adequate affinity for most purposes.
The cleavage domain from FokI has to dimerize to be active as a nuclease. The dimer interface is quite weak – the protein is essentially never seen as a dimer in solution. This turns out to be very beneficial, as a monomeric ZFN is not active; the cleavage reagent is assembled at the dual target site by binding of separate molecules, each of which must recognize its binding site. The best way to enforce cleavage by ZFNs is to bind two sets of ZFs, each linked to a monomeric cleavage domain, to neighboring sites. At the resulting high local concentration, dimerization occurs readily. Like natural FokI, ZFN pairs make staggered breaks in the two DNA strands, leaving 4-nucleotide 5’ overlaps. There appear to be no constraints on the sequence of the actual cut site.
Early studies on the ZFNs showed that they could cut DNA both in vitro and in vivo. An investigation of the relationship between the linker joining the domains of the protein and the spacer between binding sites in the DNA showed optimal cleavage in vivo when the linker was very short (L = 0) and the spacer was exactly 6 bp. A subsequent study achieved very good cleavage with the same size linker and a 5-bp spacer.
ZFNs have been shown to be functional and efficient at cleaving synthetic and genomic targets in Drosophila, mammalian cells, C. elegans, Arabidopsis, cultured plant cells, and Xenopus oocytes. Because double-strand breaks in DNA stimulate both inaccurate repair and homologous recombination in essentially all organisms, the ZFN procedures should be very broadly applicable. One issue that remains to be addressed is the apparent toxicity of some ZFNs. In Drosophila, we showed that this is due to excessive cleavage, presumably due to binding and dimerization of the ZF set at non-canonical sequences. A number of groups are working to reduce these adverse off-target effects.
In addition to linkage to a nuclease domain, zinc fingers have been used to direct other functional domains to specific sites in DNA. Several labs have made synthetic transcription factors – both activators and repressors – based on zinc fingers, and DNA methylase has been targeted in a similar fashion.
Designing ZFNs for Novel Targets
In collaboration with David Segal, we have very recently assembled a detailed protocol for making ZFNs with new finger combinations [Carroll et al., Nature Protocols 1: 1329-1341 (2006)]. The basic steps are these:
Obviously the first step reflects the interests and intentions of each individual researcher. The search for plausible ZFN sites (step 2) is greatly aided by the advent of two recent websites: www.zincfingertools.org at the Barbas Lab, and www.zincfingers.org that leads to the Voytas Lab. Each facilitates a search for sites of the form (XXX)3N6(XXX)3, where XXX are triplets for which zinc fingers exist and N is any nucleotide. Part of the challenge is to focus on “good” fingers. Essentially all of the triplets of the form 5’-GNN-3’ have corresponding fingers that have been tested extensively. Fingers for some other triplets look like they show good specificity when assayed on naked DNA, and some of these have been tested for binding in vivo. Nonetheless, only a small number of fingers has been examined in ZFNs in vivo.
When we design new ZFNs, we emphasize GNN-directed fingers – e.g., by searching for sites of the form (XXC)3N6(GXX)3. In addition, we have produced a table that rates zinc fingers that have been characterized by in vitro binding assays. Use of this table allows one to limit the ultimate choice to fingers that look most promising. It is worth reiterating, however, that relatively few fingers and very few of the possible combinations have actually been tested.
In designing coding sequences for the new finger sets, it is important to note that the N-terminal finger binds the 3’-most DNA triplet, as things are normally drawn. We usually make new finger sets from long, synthetic oligos; and two schemes are outlined in our protocol paper. One can also do oligo-directed mutagenesis on pre-existing clones, or make use of a library of individual cloned fingers made available by the Zinc Finger Consortium (www.zincfingers.org). Another feature of this design is the choice of protein framework in which to place the specificity-determining residues. Success has been achieved with frameworks from mouse Zif268, human Sp1, and the Sp1-related consensus called Sp1C.
We currently clone new ZF coding sequences in frame with the FokI cleavage domain in a pENTR vector (step 4), which facilitates transfer to ultimate expression vectors (step 5), as long as they are available in pDEST formats. Expression systems for protein production in E. coli (for in vitro testing), in Drosophila, in C. elegans, and for mRNA synthesis in vitro, are available from us. Others have achieved expression in plants and in mammalian cells.
ZFN references
Original research articles
Kim, Y.G., Cha, J. and Chandrasegaran, S. (1996). Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain. Proc. Natl. Acad. Sci USA 93: 1156-1160.
Huang, B., Schaeffer, C.J., Li, Q. and Tsai, M.D. (1996). Sp1ase: a new class IIS zinc-finger restriction endonuclease with specificity for Sp1 binding sites. J. Protein Chem. 15: 481-489.
Kim, Y.G., Shi, Y., Berg, J.M. and Chandrasegaran, S. (1997). Site-specific cleavage of DNA-RNA hybrids by zinc finger/FokI cleavage domain fusions. Gene 203: 43-49.
Smith, J., Berg, J.M. and Chandrasegaran, S. (1999). A detailed study of the substrate specificity of a chimeric restriction enzyme. Nucleic Acid Res. 27: 674-681.
Smith, J., Bibikova, M., Whitby, F.G., Reddy, A.R., Chandrasegaran, S. and Carroll, D. (2000). Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res. 28: 3361-3369.
Bibikova, M., Carroll, D., Segal, D.J., Trautman, J.K., Smith, J., Kim, Y.-G. and Chandrasegaran, S. (2001). Stimulation of homologous recombination through targeted cleavage by a chimeric nuclease. Mol. Cell. Biol. 21, 289-297.
Bibikova, M., Golic, M., Golic, K.G. and Carroll, D. (2002). Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161: 1169-1175.
Porteus, M.H. and Baltimore, D. (2003). Chimeric nucleases stimulate gene targeting in human cells. Science 300: 763.
Bibikova, M., Beumer, K., Trautman, J.K. and Carroll, D. (2003). Enhancing gene targeting with designed zinc-finger nucleases. Science 300: 764.
Urnov, F., Miller, J.C., Lee, Y.L., Beausejour, C.M., Rock, J.M., Augustus, S., Jamieson, A.C., Porteus, M.H., Gregory, P.D. and Holmes, M.C. (2005). Highly efficient human gene correction using designed zinc-finger nucleases. Nature 435: 646-651.
Lloyd, A., Plaisier, C.L., Carroll, D. and Drews, G.N. (2005). Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. Proc. Natl. Acad. Sci. USA 102: 2232-2237.
Alwin, S., Gere, M.B., Guhl, E., Effertz, K., Barbas, C.F. III, Segal, D.J., Weitzman, M.D. and Cathomen, T. (2005). Custom zinc-finger nucleases for use in human cells. Mol. Therapy 12: 610-617.
Nakatsukasa, T., Shiraishi, Y., Negi, S., Imanishi, M., Futaki, S. and Sugiura, Y. (2005). Site-specific DNA cleavage by artificial zinc finger-type nuclease with cerium-binding peptide. Biochem. Biophys. Res. Commun. 330: 247-252.
Mani, M., Smith, J., Kandavelou, K., Berg, J.M. and Chandrasegaran, S. (2005). Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage. Biochem. Biophys. Res. Commun. 334: 1191-1197.
Mani, M., Kandavelou, K., Dy, F.J., Durai, S. and Chandrasegaran, S. (2005) Design, engineering and characterization of zinc finger nucleases. Biochem. Biophys. Res. Commun. 335: 447-457.
Wright, D.A., Townsend, J.A., Winfrey, R.J. Jr., Irwin, P.A., Rajagopal, J., Lonosky, P.M., Hall, B.D., Jondle, M.D. and Voytas, D.F. (2005). High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J. 44: 693-705.
Beumer, K., Bhattacharrya, G., Bibikova, M., Trautman, J.K. and Carroll, D. (2006). Efficient gene targeting in Drosophila with zinc finger nucleases. Genetics 172: 2391-2403.
Porteus, M.H. (2006). Mammalian gene targeting with designed zinc finger nucleases. Mol. Therapy 13: 438-446.
Morton, J., Davis, M.W., Jorgensen, E.M. and Carroll, D. (2006). Induction and repair of zinc-finger nuclease-targeted double-strand breaks in C. elegans somatic cells. Proc. Natl. Acad. Sci. USA, 103: 16370-16375.
Moehle, E.A., Rock, J.M., Yee, Y.L. Jouvenot, Y., DeKelver, R.C., Gregory, P.D., Urnov, F.D. and Holmes, M.C. (2007). Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc. Natl. Acad. Sci. USA, 104: 3055-3060.
Reviews
Chandrasegaran, S, and Smith, J. (1999). Chimeric restriction enzymes: what is next? Biol. Chem. 380: 841-848.
Carroll, D. (2004). Using nucleases to stimulate homologous recombination. In Methods in Molecular Biology, vol. 262, Genetic Recombination, A.S. Waldman, ed., Humana Press, Totowa, NJ, pp. 195-207.
Porteus, M.H. and Carroll, D. (2005). Gene targeting using zinc finger nucleases. Nature Biotechnol. 23: 967-973.
Durai, S., Mani, M., Kandavelou, K. Wu, J., Porteus, M.H. and Chandrasegaran, S. (2005). Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res. 33: 5978-5990.
Kolb, A.F., Coates, C.J., Kaminski, J.M., Summers, J.B., Miller, A.D. and Segal, D.J. (2005). Site-directed genome modification: nucleic acid and protein modules for targeted integration and gene correction. Trends Biotechnol. 23: 399-406.
Klug, A. (2005). Towards therapeutic applications of engineered zinc finger proteins. FEBS Lett. 579: 892-894.
Papworth, M., Kolasinska, P. and Minczuk, M. (2006). Designer zinc-finger proteins and their applications. Gene 366: 27-38.
Dhanasekaran, M., Negi, S. and Sugiura, Y. (2006). Designer zinc finger proteins: tools for creating artificial DNA-binding functional proteins. Acc. Chem. Res. 39: 45-52.
News and Views
Wilson, J.H. (2003). Pointing fingers at the limiting step in gene targeting. Nature Biotechnol. 21: 759-760.
Marshall, A. (2003). Grasping gene targeting. Nature Biotechnol. 21: 633.
Porter, A.C.G. (2005). Two hands make light work of gene modification. Rejuvenation Res. 8: 211-215.
Kaiser, J. (2005). Putting the fingers on gene repair. Science 310: 1894-1896.
Kandavelou, K., Mani, M., Durai, S. and Chandrasegaran, S. (2005). “Magic” scissors for genome surgery. Nature Biotechnol. 23: 686-687.
High, K.A. (2005). Gene therapy: the moving finger. Nature 435: 577-579.
Scott, C.T. (2005). The zinc finger nuclease monopoly. Nature Biotechnol. 23: 915-918.
Eisenstein, M. (2005). Human gene repair at your fingertips. Nature Methods 2: 405.
Cathomen, T. and Weitzman, M.D. (2005). Pointing the finger at genetic disease. Gene Therapy 12: 1415-1416.
Other stuff
Carroll, D., Morton, J.J., Beumer, K.J. and Segal, D.J. (2006). Design, construction and in vitro testing of zinc finger nucleases. Nature Protocols 1: 1329-1341.
Wright, D. A., Thibodeau-Beganny, S., Sander, J. D., Wiinfrey, R. J., Hirsh, A. S., Eichtinger, M., Fu, F., Porteus, M. H., Dobbs, D., Voytas, D. F., and Joung, J. K. (2006). Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nature Protocols 1: 1637-1652.
Mandell, J.G. and Barbas, C.F. III (2006). Zinc finger tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res. 34: W516-523. (www.zincfingertools.org)