An improved zinc-finger nuclease architecture for highly specific genome editing

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 catalytic...

Full description

Saved in:
Bibliographic Details
Published in:Nature biotechnology 2007-07, Vol.25 (7), p.778-785
Main Authors: Wang, Jianbin, Rebar, Edward J, Guschin, Dmitry Y, Lee, Ya-Li, Holmes, Michael C, Miller, Jeffrey C, Gregory, Philip D, Kim, Kenneth A, Waite, Adam J, Beausejour, Christian M, Wang, Nathaniel S, Pabo, Carl O, Rupniewski, Igor
Format: Article
Language:English
Subjects:
Agriculture
Base Sequence
Binding Sites
Bioinformatics
Biological and medical sciences
Biomedical and Life Sciences
Biomedical Engineering/Biotechnology
Biomedicine
Biotechnology
Biotechnology - methods
Catalysis
Deoxyribonucleases, Type II Site-Specific - chemistry
Deoxyribonucleic acid
Dimerization
DNA
Fundamental and applied biological sciences. Psychology
Genome
Genomics
Green Fluorescent Proteins - chemistry
Humans
Innovations
K562 Cells
Life Sciences
Methods. Procedures. Technologies
Models, Biological
Molecular Conformation
Molecular Sequence Data
Protein engineering
Protein Structure, Tertiary
Reagents
Technological change
Zinc
Zinc Fingers
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:1087-0156
1546-1696
DOI:10.1038/nbt1319