Abstract
The technical advances in developing artificial endonucleases, such as zinc finger nucleases (ZFNs), have opened a wide field of applications in the genome engineering arena, including the therapeutic correction of mutated genes in the human genome. Gene editing frequencies of up to 50% in human cells under non-selective conditions reveal the power of the ZFN technology. Activity and toxicity of ZFNs are determined by a number of parameters, including the specificity of DNA binding, the kinetics of dimerization of the two ZFN subunits, and the catalytic activity. In order to investigate these parameters individually, a cell-free system that models these reactions is essential. Here, we present a simple and fast method for the functional testing of ZFNs in vitro.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Carroll, D. (2008) Progress and prospects: zinc-finger nucleases as gene therapy agents. Gene Ther. 15, 1463–1468.
Cathomen, T. and Joung, J.K. (2008) Zinc-finger nucleases: the next generation emerges. Mol Ther. 16, 1200–1207.
Vasquez, K.M., Marburger, K., Intody, Z., and Wilson, J.H. (2001) Manipulating the mammalian genome by homologous recombination. Proc Natl Acad Sci USA. 98, 8403–8410.
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., Unger-Wallace, E., Sander, J.D., Muller-Lerch, F., Fu, F., Pearlberg, J., Gobel, C., Dassie, J.P., Pruett-Miller, S.M., Porteus, M.H., Sgroi, D.C., Iafrate, A.J., Dobbs, D., McCray, P.B., Jr., Cathomen, T., Voytas, D.F., and Joung, J.K. (2008) Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell. 31, 294–301.
Lombardo, A., Genovese, P., Beausejour, C.M., Colleoni, S., Lee, Y.L., Kim, K.A., Ando, D., Urnov, F.D., Galli, C., Gregory, P.D., Holmes, M.C., and Naldini, L. (2007) Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol. 25, 1298–1306.
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., and June, C.H. (2008) Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol. 26, 808–816.
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., and Amacher, S.L. (2008) Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol. 26, 702–708.
Foley, J.E., Yeh, J.R., Maeder, M.L., Reyon, D., Sander, J.D., Peterson, R.T., and Joung, J.K. (2009) Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN). PLoS One. 4, e4348.
Meng, X., Noyes, M.B., Zhu, L.J., Lawson, N.D., and Wolfe, S.A. (2008) Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol. 26, 695–701.
Morton, J., Davis, M.W., Jorgensen, E.M., and Carroll, D. (2006) Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells. Proc Natl Acad Sci USA. 103, 16370–16375.
Bibikova, M., Beumer, K., Trautman, J.K., and Carroll, D. (2003) Enhancing gene targeting with designed zinc finger nucleases. Science. 300, 764.
Kim, Y.G., Cha, J., and Chandrasegaran, S. (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA. 93, 1156–1160.
Elrod-Erickson, M., Rould, M.A., Nekludova, L., and Pabo, C.O. (1996) Zif268 protein-DNA complex refined at 1.6 A: a model system for understanding zinc finger-DNA interactions. Structure. 4, 1171–1180.
Pavletich, N.P. and Pabo, C.O. (1991) Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science. 252, 809–817.
Dreier, B., Segal, D.J., and Barbas, C.F., 3rd (2000) Insights into the molecular recognition of the 5ʹ-GNN-3ʹ family of DNA sequences by zinc finger domains. J Mol Biol. 303, 489–502.
Beerli, R.R. and Barbas, C.F., 3rd (2002) Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol. 20, 135–141.
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 chimeric nucleases. Mol Cell Biol. 21, 289–297.
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.
Miller, J.C., Holmes, M.C., Wang, J., Guschin, D.Y., Lee, Y.L., Rupniewski, I., Beausejour, C.M., Waite, A.J., Wang, N.S., Kim, K.A., Gregory, P.D., Pabo, C.O., and Rebar, E.J. (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol. 25, 778–785.
Szczepek, M., Brondani, V., Buchel, J., Serrano, L., Segal, D.J., and Cathomen, T. (2007) Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol. 25, 786–793.
Alwin, S., Gere, M.B., Guhl, E., Effertz, K., Barbas, C.F., 3rd, Segal, D.J., Weitzman, M.D., and Cathomen, T. (2005) Custom zinc-finger nucleases for use in human cells. Mol Ther. 12, 610–617.
Cornu, T.I., Thibodeau-Beganny, S., Guhl, E., Alwin, S., Eichtinger, M., Joung, J.K., and Cathomen, T. (2008) DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases. Mol Ther. 16, 352–358.
Handel, E.M., Alwin, S., and Cathomen, T. (2009) Expanding or restricting the target site repertoire of zinc-finger nucleases: the inter-domain linker as a major determinant of target site selectivity. Mol Ther. 17, 104–111.
Isalan, M., Choo, Y., and Klug, A. (1997) Synergy between adjacent zinc fingers in sequence-specific DNA recognition. Proc Natl Acad Sci USA. 94, 5617–5621.
Mandell, J.G. and Barbas, C.F., 3rd (2006) Zinc finger tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res. 34, W516–W523.
Acknowledgments
The authors thank Anton McCaffrey and Thomas J. Cradick for sharing their know-how on ZFN in vitro cleavage reactions and Tatjana Cornu for carefully reading this manuscript. This chapter is based on work supported by grant LSHB-CT2006-037783 ZNIP of the European Commission’s 6th Framework Programme.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Cathomen, T., Şöllü, C. (2010). In Vitro Assessment of Zinc Finger Nuclease Activity. In: Mackay, J., Segal, D. (eds) Engineered Zinc Finger Proteins. Methods in Molecular Biology, vol 649. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-753-2_13
Download citation
DOI: https://doi.org/10.1007/978-1-60761-753-2_13
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-752-5
Online ISBN: 978-1-60761-753-2
eBook Packages: Springer Protocols