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Design, construction and in vitro testing of zinc finger nucleases

Abstract

Zinc finger nucleases (ZFNs) are hybrid proteins that have been developed as targetable cleavage reagents for double-stranded DNA, both in vitro and in vivo. This protocol describes the design and construction of new DNA-binding domains comprised of zinc fingers (ZFs) directed at selected DNA sequences. Because the ZFNs must dimerize to cut DNA, they are designed in pairs for any new site. The first step is choosing a DNA segment of interest and searching it for sequences that can be recognized by combinations of existing ZFs. The second step is the construction of coding sequences for the selected ZF sets. Third, these coding sequences are linked to that of the nonspecific cleavage domain from the FokI restriction endonuclease in a cloning vector of choice. Finally, the ZFNs are expressed in Escherichia coli, partially purified, and tested in vitro for cleavage of the target sequences to which they were designed. If all goes smoothly, design, construction and cloning can be completed in about two weeks, with expression and testing completed in one additional week.

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Figure 1: Diagram of a pair of ZFNs bound to DNA.
Figure 2: Scheme of oligonucleotide-based strategies for synthesizing zinc finger coding sequences.
Figure 3: Illustration of the scheme to produce a four-finger ZFN clone from three-finger and one-finger fragments produced by the seven-primer strategy.
Figure 4: Some of the potential ZFN targets identified in the D. melanogaster ry gene.
Figure 5: Specificity-determining residues for each of the triplets in the selected site in the ry gene (Fig.4) picked from Table 1.
Figure 6: Coding sequences for the ryA ZFN in the Zif268 framework.
Figure 7: PCR products from a long-primer strategy.
Figure 8: The seven-primer scheme for synthesis of the ZF coding region in the SP1C framework.
Figure 9: PCR products from the seven-primer strategy.
Figure 10: Results of a typical colony PCR.
Figure 11: Typical SDS-PAGE analysis of ZFN purification.
Figure 12: Example of an in vitro assay of ZFN activity.

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Acknowledgements

We are grateful to M. Bibikova for establishing many of these procedures and to members of the Carroll Lab who have used them. R. Watkins produced the data shown in Figure 7. We acknowledge S. Chandrasegaran, who produced the first ZFNs and with whom we collaborated on early characterizations of them. We also acknowledge C. Barbas for his many contributions, including most of the modified zinc finger domains described here, and we thank him for allowing us to cite his website. This work was supported in part by research grants from the US National Institutes of Health to D.C. (GM58504 and GM65173) and to D.J.S. (CA103651).

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Correspondence to Dana Carroll or David J Segal.

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Carroll, D., Morton, J., Beumer, K. et al. Design, construction and in vitro testing of zinc finger nucleases. Nat Protoc 1, 1329–1341 (2006). https://doi.org/10.1038/nprot.2006.231

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