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 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.
Miller
Jeffrey C
JC
Sangamo BioSciences, Inc., Pt. Richmond Tech Center, 501 Canal Blvd., Suite A100 Richmond, California 94804, USA.
Holmes
Michael C
MC
Wang
Jianbin
J
Guschin
Dmitry Y
DY
Lee
Ya-Li
YL
Rupniewski
Igor
I
Beausejour
Christian M
CM
Waite
Adam J
AJ
Wang
Nathaniel S
NS
Kim
Kenneth A
KA
Gregory
Philip D
PD
Pabo
Carl O
CO
Rebar
Edward J
EJ
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
D049452:Green Fluorescent Proteins; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific