An improved zinc-finger nuclease architecture for highly specific genome editing
Abstract
Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.
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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 Miller et al., 2007
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- Authors
- Miller, J. C., Holmes, M. C., Wang, J., Guschin, D. Y., Lee, Y.-L., Rupniewski, I., Beausejour, C. M., Waite, A. J., Wang, N. S., Kim, K. A., Gregory, P. D., Pabo, C. O., & Rebar, E. J.
- Affiliations
- Sangamo BioSciences (United States) (US)
- Harvard University (US)
- Corresponding author
- Rebar, Edward J
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2007-07
- Volume
- 25
- Issue
- 7
- Pages
- 778-785
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- OpenAlex
- W2116565356
- Cited by
- 1,051
- References
- 5
- Citations by year
- 2026: 13 · 2025: 25 · 2024: 40 · 2023: 34 · 2022: 48 · 2021: 47 · 2020: 60 · 2019: 39 · 2018: 65 · 2017: 63 · 2016: 83 · 2015: 85 · 2014: 102 · 2013: 72 · 2012: 72
- FWCI
- 18.03
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- zinc finger nucleases, genome editing, heterodimerization, off-target cleavage, structure-based design, DNA cleavage specificity, DNA double-strand breaks
- MeSH terms
- Base Sequence, Binding Sites, Biotechnology, Catalysis, Humans, Models, Biological, Molecular Conformation, Molecular Sequence Data, Deoxyribonucleases, Type II Site-Specific, Zinc Fingers, Genome, Protein Structure, Tertiary, Dimerization, K562 Cells, Green Fluorescent Proteins
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