Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection
Open accessAbstract
Engineered zinc-finger nucleases (ZFNs) are promising tools for genome manipulation, and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 10(11) DNA sequences for cleavage by active, dimeric ZFNs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs: CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGFA genes, respectively. Analysis of identified sites in one cultured human cell line revealed CCR5-224-induced changes at nine off-target loci, though this remains to be tested in other relevant cell types. Similarly, we observed 31 off-target sites cleaved by VF2468 in cultured human cells. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future ZFN design.
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Proper evaluation of the specificity of any particular ZFN pair will require the use of the relevant cell type, the intended ZFN dose and the delivery method for that specific application.
“Taken together, these studies highlight the fact that proper evaluation of the specificity of any particular ZFN pair will require the use of the relevant cell type, the intended ZFN dose and the delivery method for that specific application.”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [64]
Pattanayak et al. found that high-affinity binding of one CCR5-specific ZFN monomer in a pair can compensate for another monomer's weak binding.
“In an interesting study, Pattanayak et al. found that high-affinity binding of one CCR5-specific ZFN monomer in a pair can compensate for another monomer's weak binding [64].”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [64]
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- Authors
- Pattanayak, V., Ramirez, C. L., Joung, J. K., & Liu, D. R.
- Affiliations
- Howard Hughes Medical Institute (US)
- Harvard University (US)
- Corresponding author
- Liu, David R
- Venue
- Nature Methods
- Published
- 2011-08-07
- Volume
- 8
- Issue
- 9
- Pages
- 765-770
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- PMC
- PMC3164905
- OpenAlex
- W2137409670
- Cited by
- 453
- References
- 34
- Citations by year
- 2026: 2 · 2025: 10 · 2024: 20 · 2023: 14 · 2022: 13 · 2021: 28 · 2020: 19 · 2019: 13 · 2018: 23 · 2017: 24 · 2016: 51 · 2015: 46 · 2014: 61 · 2013: 63 · 2012: 48
- FWCI
- 24.56
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Methods (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Howard Hughes Medical Institute
- National Institutes of Health (DP1OD006862)
- National Institute of General Medical Sciences (DP1GM105378, R01GM088040, R01GM065400)
- National Institute of Neurological Disorders and Stroke (P30NS045776)
- 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, off-target cleavage, cleavage specificity, in vitro selection, CCR5, VEGF-A, genome editing, binding energy, human genome
- MeSH terms
- Base Sequence, Binding Sites, Cells, Cultured, DNA, DNA-Binding Proteins, Endodeoxyribonucleases, Humans, Substrate Specificity, Zinc Fingers, K562 Cells
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