title: 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.
authors.affiliation: Sangamo BioSciences, Inc., Pt. Richmond Tech Center, 501 Canal Blvd., Suite A100 Richmond, California 94804, USA.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Miller
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.forename: Jianbin
authors.initials: J
authors.lastname: Wang
authors.forename: Dmitry Y
authors.initials: DY
authors.lastname: Guschin
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Lee
authors.forename: Igor
authors.initials: I
authors.lastname: Rupniewski
authors.forename: Christian M
authors.initials: CM
authors.lastname: Beausejour
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Waite
authors.forename: Nathaniel S
authors.initials: NS
authors.lastname: Wang
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Kim
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Carl O
authors.initials: CO
authors.lastname: Pabo
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Rebar
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D001709:Biotechnology / Q000379:methods*; D002384:Catalysis; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D019281:Dimerization; D016678:Genome; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D020014:K562 Cells; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016335:Zinc Fingers
chemical_list: D049452:Green Fluorescent Proteins; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific