Abstract
Customized zinc-finger nucleases (ZFNs) have developed into a promising technology to precisely alter mammalian genomes for biomedical research, biotechnology, or human gene therapy. In the context of synthetic biology, the targeted integration of a transgene or reporter cassette into a “neutral site” of the human genome, such as the AAVS1 locus, permits the generation of isogenic human cell lines with two major advantages over standard genetic manipulation techniques: minimal integration site-dependent effects on the transgene and, vice versa, no functional perturbation of the host-cell transcriptome. Here we describe in detail how ZFNs can be employed to target integration of a transgene cassette into the AAVS1 locus and how to characterize the targeted cells by PCR-based genotyping.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Capecchi, M. R. (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century, Nat Rev Genet 6, 507–512.
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.
Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., and Gregory, P. D. (2010) Genome editing with engineered zinc finger nucleases, Nat Rev Genet 11, 636–646.
Jasin, M. (1996) Genetic manipulation of genomes with rare-cutting endonucleases, Trends Genet 12, 224–228.
Cathomen, T., and Joung, K. J. (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.
Kim, Y. G., Cha, J., and Chandrasegaran, S. (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain, Proc Natl Acad Sci U S A 93, 1156–1160.
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.
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.
Kotin, R. M., Linden, R. M., and Berns, K. I. (1992) Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination, Embo J 11, 5071–5078.
Ogata, T., Kozuka, T., and Kanda, T. (2003) Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA, J Virol 77, 9000–9007.
Smith, J. R., Maguire, S., Davis, L. A., Alexander, M., Yang, F., Chandran, S., ffrench-Constant, C., and Pedersen, R. A. (2008) Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration, Stem Cells 26, 496–504.
Hockemeyer, D., Soldner, F., Beard, C., Gao, Q., Mitalipova, M., DeKelver, R. C., Katibah, G. E., Amora, R., Boydston, E. A., Zeitler, B., Meng, X., Miller, J. C., Zhang, L., Rebar, E. J., Gregory, P. D., Urnov, F. D., and Jaenisch, R. (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases, Nat Biotechnol 27, 851–857.
DeKelver, R. C., Choi, V. M., Moehle, E. A., Paschon, D. E., Hockemeyer, D., Meijsing, S. H., Sancak, Y., Cui, X., Steine, E. J., Miller, J. C., Tam, P., Bartsevich, V. V., Meng, X., Rupniewski, I., Gopalan, S. M., Sun, H. C., Pitz, K. J., Rock, J. M., Zhang, L., Davis, G. D., Rebar, E. J., Cheeseman, I. M., Yamamoto, K. R., Sabatini, D. M., Jaenisch, R., Gregory, P. D., and Urnov, F. D. (2010) Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome, Genome Res 20, 1133–1142.
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.
Söllü, C., Pars, K., Cornu, T. I., Thibodeau-Beganny, S., Maeder, M. L., Joung, K. J., Heilbronn, R., and Cathomen, T. (2010) Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion, Nucleic Acids Res 38, 8269–8276.
Zou, J., Maeder, M. L., Mali, P., Pruett-Miller, S. M., Thibodeau-Beganny, S., Chou, B. K., Chen, G., Ye, Z., Park, I. H., Daley, G. Q., Porteus, M. H., Joung, J. K., and Cheng, L. (2009) Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells, Cell Stem Cell 5, 97–110.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Dreyer, AK., Cathomen, T. (2012). Zinc-Finger Nucleases-Based Genome Engineering to Generate Isogenic Human Cell Lines. In: Weber, W., Fussenegger, M. (eds) Synthetic Gene Networks. Methods in Molecular Biology, vol 813. Humana Press. https://doi.org/10.1007/978-1-61779-412-4_8
Download citation
DOI: https://doi.org/10.1007/978-1-61779-412-4_8
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-411-7
Online ISBN: 978-1-61779-412-4
eBook Packages: Springer Protocols