Cited by 453CRISPR and Genetic EngineeringRNA Interference and Gene DeliveryRNA regulation and disease

Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection

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Pattanayak, Vikram, Ramirez, Cherie L, Joung, J Keith, Liu, David R·2011·Nature Methods

Abstract

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
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
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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