Custom zinc-finger nucleases for use in human cells

Stephen Alwin, Maja B. Gere, Eva Guhl, Karin Effertz, Carlos F. Barbas, David Segal, Matthew D. Weitzman, Toni Cathomen

Research output: Contribution to journalArticle

129 Citations (Scopus)

Abstract

Genome engineering through homologous recombination (HR) is a powerful instrument for studying biological pathways or creating treatment options for genetic disorders. In mammalian cells HR is rare but the creation of targeted DNA double-strand breaks stimulates HR significantly. Here, we present a method to generate, evaluate, and optimize rationally designed endonucleases that promote HR. The DNA-binding domains were synthesized by assembling predefined zinc-finger modules selected by phage display. Attachment of a transcriptional activation domain allowed assessment of DNA binding in reporter assays, while fusion with an endonuclease domain created custom nucleases that were tested for their ability to stimulate HR in episomal and chromosomal gene repair assays. We demonstrate that specificity, expression kinetics, and protein design are crucial parameters for efficient gene repair and that our two-step assay allows one to go quickly from design to testing to successful employment of the custom nucleases in human cells.

Original languageEnglish (US)
Pages (from-to)610-617
Number of pages8
JournalMolecular Therapy
Volume12
Issue number4
DOIs
StatePublished - Oct 2005
Externally publishedYes

Fingerprint

Homologous Recombination
Zinc Fingers
Endonucleases
Inborn Genetic Diseases
Double-Stranded DNA Breaks
DNA
Bacteriophages
Transcriptional Activation
Genes
Genome
Proteins

Keywords

  • Artificial endonucleases
  • Chimeric nucleases
  • Gene correction
  • Site-directed recombination
  • Zinc-Finger

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Alwin, S., Gere, M. B., Guhl, E., Effertz, K., Barbas, C. F., Segal, D., ... Cathomen, T. (2005). Custom zinc-finger nucleases for use in human cells. Molecular Therapy, 12(4), 610-617. https://doi.org/10.1016/j.ymthe.2005.06.094

Custom zinc-finger nucleases for use in human cells. / Alwin, Stephen; Gere, Maja B.; Guhl, Eva; Effertz, Karin; Barbas, Carlos F.; Segal, David; Weitzman, Matthew D.; Cathomen, Toni.

In: Molecular Therapy, Vol. 12, No. 4, 10.2005, p. 610-617.

Research output: Contribution to journalArticle

Alwin, S, Gere, MB, Guhl, E, Effertz, K, Barbas, CF, Segal, D, Weitzman, MD & Cathomen, T 2005, 'Custom zinc-finger nucleases for use in human cells', Molecular Therapy, vol. 12, no. 4, pp. 610-617. https://doi.org/10.1016/j.ymthe.2005.06.094
Alwin S, Gere MB, Guhl E, Effertz K, Barbas CF, Segal D et al. Custom zinc-finger nucleases for use in human cells. Molecular Therapy. 2005 Oct;12(4):610-617. https://doi.org/10.1016/j.ymthe.2005.06.094
Alwin, Stephen ; Gere, Maja B. ; Guhl, Eva ; Effertz, Karin ; Barbas, Carlos F. ; Segal, David ; Weitzman, Matthew D. ; Cathomen, Toni. / Custom zinc-finger nucleases for use in human cells. In: Molecular Therapy. 2005 ; Vol. 12, No. 4. pp. 610-617.
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