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Gene targeting using zinc finger nucleases

Nature Biotechnology volume 23, pages 967–973 (2005)Cite this article

Abstract

The ability to achieve site-specific manipulation of the mammalian genome has widespread implications for basic and applied research. Gene targeting is a process in which a DNA molecule introduced into a cell replaces the corresponding chromosomal segment by homologous recombination, and thus presents a precise way to manipulate the genome. In the past, the application of gene targeting to mammalian cells has been limited by its low efficiency. Zinc finger nucleases (ZFNs) show promise in improving the efficiency of gene targeting by introducing DNA double-strand breaks in target genes, which then stimulate the cell's endogenous homologous recombination machinery. Recent results have shown that ZFNs can be used to create targeting frequencies of up to 20% in a human disease-causing gene. Future work will be needed to translate these in vitro findings to in vivo applications and to determine whether zinc finger nucleases create undesired genomic instability.

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Figure 1: Double-strand break repair.

Bob Crimi

Figure 2: ZFN homodimer binding to DNA. Shown is a three-finger zinc finger linked to the Fn domain through a flexible peptide linker.
Figure 3: GFP gene-targeting reporter system.

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Acknowledgements

We thank Scott Cameron, Jim Amatruda, Brian Cauff, Shondra Pruett, Patrick Connelly, Michael Holmes and Philip Gregory for reading the manuscript and for their helpful comments. The work in the Porteus lab is supported by the Burroughs-Wellcome Fund, a K08 award from the National Heart and Blood Institute, and UT Southwestern Medical Center. Work in the Carroll lab is supported by research grants from the US Public Health Service.

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  1. Departments of Pediatrics and Biochemistry, University of Texas Southwestern Medical Center,

    Matthew H Porteus

  2. Department of Biochemistry, University of Utah, School of Medicine,

    Dana Carroll

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Correspondence to Matthew H Porteus.

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D.C. is an inventor on US and international patent applications that describe the ZFN-based gene-targeting technology.

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Porteus, M., Carroll, D. Gene targeting using zinc finger nucleases. Nat Biotechnol 23, 967–973 (2005). https://doi.org/10.1038/nbt1125

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