Abstract
The recent development of artificial zinc finger nucleases (ZFNs) for targeted genome editing has opened a broad range of possibilities in biotechnology and gene therapy. The ZFN technology allows a researcher to deliberately choose a target site in a complex genome and create appropriate nucleases to insert a DNA double-strand break (DSB) at that site. Gene editing frequencies of up to 50% in non-selected human cells attest to the power of this technology. Potential side effects of applying ZFNs include toxicity due to cleavage at off-target sites. This can be brought about by insufficient specificity of DNA binding, hence allowing ZFN activity at similar target sequences within the genome, or by activation of the ZFN nuclease domains before the nuclease is properly bound to the DNA. Here, we describe two different methods to quantify ZFN-associated toxicity: the genotoxicity assay is based on quantification of DSB repair foci induced by ZFNs whereas the cytotoxicity is based on assessing cell survival after application of ZFNs.
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
Segal, D.J., Cathomen, T., and Joung, J.K. (2008) Zinc-finger nucleases: the next generation emerges. Mol Ther. 16, 1200–1207.
Carroll, D. (2008) Progress and prospects: zinc-finger nucleases as gene therapy agents. Gene Ther. 15, 1463–1468.
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.
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.
Bitinaite, J., Wah, D.A., Aggarwal, A.K., and Schildkraut, I. (1998) FokI dimerization is required for DNA cleavage. Proc Natl Acad Sci USA. 95, 10570–10575.
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.
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.
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.
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.
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.
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.
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.
Pruett-Miller, S.M., Connelly, J.P., Maeder, M.L., Joung, J.K., and Porteus, M.H. (2008) Comparison of zinc finger nucleases for use in gene targeting in mammalian cells. Mol Ther. 16, 707–717.
Porteus, M.H. and Baltimore, D. (2003) Chimeric nucleases stimulate gene targeting in human cells. Science. 300, 763.
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.
Rogakou, E.P., Boon, C., Redon, C., and Bonner, W.M. (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol. 146, 905–916.
Acknowledgments
We thank Cem Şöllü for careful reading of the chapter. This chapter is based on work supported by grant CA 311/2 of the Research Priority Programme 1230 (SPP 1230) of the German Research Foundation (DFG).
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
Cornu, T.I., Cathomen, T. (2010). Quantification of Zinc Finger Nuclease-Associated Toxicity. 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_14
Download citation
DOI: https://doi.org/10.1007/978-1-60761-753-2_14
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