Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases
Open accessAbstract
Artificial endonucleases consisting of a FokI cleavage domain tethered to engineered zinc-finger DNA-binding proteins have proven useful for stimulating homologous recombination in a variety of cell types. Because the catalytic domain of zinc-finger nucleases (ZFNs) must dimerize to become active, two subunits are typically assembled as heterodimers at the cleavage site. The use of ZFNs is often associated with significant cytotoxicity, presumably due to cleavage at off-target sites. Here we describe a structure-based approach to reducing off-target cleavage. Using in silico protein modeling and energy calculations, we increased the specificity of target site cleavage by preventing homodimerization and lowering the dimerization energy. Cell-based recombination assays confirmed that the modified ZFNs were as active as the original ZFNs but elicit significantly less genotoxicity. The improved safety profile may facilitate therapeutic application of the ZFN technology.
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Because the genome contains numerous sequence repetitions or highly homologous DNA sequences, efforts have been made to improve off-target effects and specificity of ZFN and TALEN enzymes.
“Because the genome contains numerous sequence repetitions or has highly homologous DNA sequences, efforts have been made in the last years to improve the offtarget effects and the specificity of these enzymes (Miller et al., 2007;Szczepek et al., 2007;Guo et al., 2010;Doyon et al., 2011).”
From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Szczepek et al., 2007
The Results (page not provided) say the authors altered ZFN dimerization to improve cleavage specificity, and the Discussion links ZFN toxicity to off-target cleavage at related DNA sites. The paper does not address TALENs or establish that numerous genomic repetitions or highly homologous sequences motivated this work. (LLM, conf=0.86)
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- Authors
- Szczepek, M., Brondani, V., Büchel, J., Serrano, L., Segal, D. J., & Cathomen, T.
- Affiliations
- Charité – Universitätsmedizin Berlin (DE)
- University of California, Davis (US)
- European Molecular Biology Laboratory (ES)
- Corresponding author
- Segal, David J
- Cathomen, Toni
- Venue
- Nature Biotechnology
- Published
- 2007-07
- Volume
- 25
- Issue
- 7
- Pages
- 786-793
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- OpenAlex
- W2080885049
- Cited by
- 501
- References
- 4
- Citations by year
- 2026: 4 · 2025: 7 · 2024: 11 · 2023: 12 · 2022: 14 · 2021: 12 · 2020: 13 · 2019: 12 · 2018: 21 · 2017: 26 · 2016: 48 · 2015: 40 · 2014: 47 · 2013: 40 · 2012: 48
- FWCI
- 13.52
- Type
- journal article
- Language
- en
- Open access
- green
- escholarship.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- FreiDok plus (Universitätsbibliothek Freiburg) (submittedVersion)
- Funders
- National Cancer Institute (CA 103651, R21CA103651)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- zinc finger nucleases, dimer interface, homodimerization, off-target cleavage, cytotoxicity, genotoxicity, homologous recombination, site-specific DNA cleavage, energy calculation
- MeSH terms
- Amino Acid Sequence, Base Sequence, Biotechnology, Cell Line, Humans, Molecular Sequence Data, Protein Conformation, Recombination, Genetic, Deoxyribonucleases, Type II Site-Specific, Zinc Fingers, Sequence Homology, Amino Acid, Codon, Terminator, Dimerization, Catalytic Domain, Cell Line, Tumor
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