Springer Protocols: Abstract: 17. Generation and Functional Analysis of Zinc Finger Nucleases

17. Generation and Functional Analysis of Zinc Finger Nucleases

By: Toni Cathomen³, David J. Segal³, Vincent Brondani³, Felix Müller-Lerch³

Abstract

Full Text

Download PDF (544K)

The recent development of artificial endonucleases with tailored specificities has opened the door for a wide range of new applications, including the correction of mutated genes directly in the chromosome. This kind of gene therapy is based on homologous recombination, which can be stimulated by the creation of a targeted DNA double-strand break (DSB) near the site of the desired recombination event. Artificial nucleases containing zinc finger DNA-binding domains have provided important proofs of concept, showing that inserting a DSB in the target locus leads to gene correction frequencies of 1–18% in human cells. In this paper, we describe how zinc finger nucleases are assembled by polymerase chain reaction (PCR) and present two methods to assess these custom nucleases quickly in vitro and in a cell-based recombination assay.

Affiliation(s): (3) Charité Medical School, Institute of Virology, Berlin, Germany

Book Title: Gene Therapy Protocols: Design and Characterization of Gene Transfer Vectors

Series: Methods in Molecular Biology | Volume: 434 | Pub. Date: Mar-07-2008 | Page Range: 277-290 | DOI: 10.1007/978-1-60327-248-3_17

Subject: Genetics/Genomics

Key Words: ZFN - artificial nuclease - custom nuclease - homologous recombination - HR - DNA double strand break - DSB

References

Download References

1.	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.

2.	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., and Holmes, M. C. (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435:646–651.

3.	Porteus, M. H. (2006) Mammalian gene targeting with designed zinc finger nucleases. Mol Ther 13:438–446.

4.	Porteus, M. H. and Baltimore, D. (2003) Chimeric nucleases stimulate gene targeting in human cells. Science 300:763.

5.	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.

6.	Beumer, K., Bhattacharyya, G., Bibikova, M., Trautman, J. K., and Carroll, D. (2006) Efficient gene targeting in Drosophila with zinc-finger nucleases. Genetics 172:2391–2403.

7.	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.

8.	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.

9.	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.

10.	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.

11.	Rebar, E. J. and Pabo, C. O. (1994) Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. Science 263:671–673.

12.	Choo, Y. and Klug, A. (1994) Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions. Proc Natl Acad Sci USA 91:11168–11172.

13.	Jamieson, A. C., Wang, H., and Kim, S. H. (1996) A zinc finger directory for high-affinity DNA recognition. Proc Natl Acad Sci USA 93:12834–12839.

14.	Wu, H., Yang, W. P., and Barbas, C. F., 3rd. (1995) Building zinc fingers by selection: toward a therapeutic application. Proc Natl Acad Sci USA 92: 344–348.

15.	Liu, Q., Xia, Z., Zhong, X., and Case, C. C. (2002) Validated zinc finger protein designs for all 16 GNN DNA triplet targets. J Biol Chem 277: 3850–3856.

16.	Segal, D. J., Dreier, B., Beerli, R. R., and Barbas, C. F., 3rd. (1999) Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5’-GNN-3’ DNA target sequences. Proc Natl Acad Sci USA 96:2758–2763.

17.	Dreier, B., Beerli, R. R., Segal, D. J., Flippin, J. D., and Barbas, C. F., 3rd. (2001) Development of zinc finger domains for recognition of the 5’-ANN-3’ family of DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem 276:29466–29478.

18.	Dreier, B., Fuller, R. P., Segal, D. J., Lund, C. V., Blancafort, P., Huber, A., Koksch, B., and Barbas, C. F., 3rd. (2005) Development of zinc finger domains for recognition of the 5’-CNN-3’ family DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem 280:35588–35597.

19.	Blancafort, P., Magnenat, L., and Barbas, C. F., 3rd. (2003) Scanning the human genome with combinatorial transcription factor libraries. Nat Biotechnol 21: 269–274.

20.	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.

21.	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.

22.	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.

23.	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:W516–523.

24.	Greisman, H. A. and Pabo, C. O. (1997) A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites. Science 275:657–661.

25.	Isalan, M., Klug, A., and Choo, Y. (2001) A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nat Biotechnol 19:656–660.

26.	Cheng, X., Boyer, J. L., and Juliano, R. L. (1997) Selection of peptides that functionally replace a zinc finger in the Sp1 transcription factor by using a yeast combinatorial library. Proc Natl Acad Sci USA 94:14120–14125.

27.	Hughes, M. D., Zhang, Z. R., Sutherland, A. J., Santos, A. F., and Hine, A. V. (2005) Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing: identification of novel zinc finger proteins. Nucleic Acids Res 33:e32.

28.	Hurt, J. A., Thibodeau, S. A., Hirsh, A. S., Pabo, C. O., and Joung, J. K. (2003) Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection. Proc Natl Acad Sci USA 100:12271–12276.

29.	Radecke, F., Peter, I., Radecke, S., Gellhaus, K., Schwarz, K., and Cathomen, T. (2006) Targeted chromosomal gene modification in human cells by single-stranded oligodeoxynucleotides in the presence of a DNA double-strand break. Mol Ther 14:798–808.

Comments (Loading...)

Post comment/View all comments