Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures

  title={Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures},
  author={Yannick Doyon and Thuy D. Vo and Matthew C. Mendel and Shon G Greenberg and Jianbin Wang and Danny F Xia and Jeffrey C. Miller and Fyodor D. Urnov and Philip D. Gregory and Michael C. Holmes},
  journal={Nature Methods},
Zinc-finger nucleases (ZFNs) drive efficient genome editing by introducing a double-strand break into the targeted gene. Cleavage is induced when two custom-designed ZFNs heterodimerize upon binding DNA to form a catalytically active nuclease complex. The importance of this dimerization event for subsequent cleavage activity has stimulated efforts to engineer the nuclease interface to prevent undesired homodimerization. Here we report the development and application of a yeast-based selection… 

Highly active zinc-finger nucleases by extended modular assembly

This first systematic study on the effect of array length on ZFN activity suggests that modular assembly ZFNs are able to target more DNA sequences with higher success rates than other current methods.

Zinc-finger recombinase activities in vitro

It is shown that purified ZFRs catalyse efficient high-specificity reciprocal recombination between pairs of Z-sites in vitro, and that the design of the ZFR protein itself is also a crucial variable affecting activity.

Origins of Programmable Nucleases for Genome Engineering.

A novel zinc-finger nuclease platform with a sequence-specific cleavage module

The ZF-PvuII platform is presented as a valid alternative to conventional ZFNs and cleave DNA at addressed sites with a >1000-fold preference over unaddressed PvuII sites in vitro as well as in cellula.

[Progress in zinc finger nuclease engineering for targeted genome modification].

The mechanism of how it works, currently available target assessment, ZFP library construction and screening methods, target modification strategies, as well as a collection of specie and genes that have been successfully modified by ZFN are focused on.

Diversifying the structure of zinc finger nucleases for high-precision genome editing

An expanded set of zinc finger nuclease architectures are developed that increase the available configurations by a factor of 64 and can target almost every base at loci of therapeutic significance and show that these new architectures may be used for targeting three loci with a high degree of precision, efficiency, and specificity.

Synthetic zinc finger nuclease design and rapid assembly.

This is the first demonstration of in silico-designed, oligonucleotide-assembled, synthetic ZFNs, requiring no specialized templates or reagents that are capable of endogenous human gene target site activity, and should facilitate a more widespread use of Z FNs in the research community.

Enhancing gene editing specificity by attenuating DNA cleavage kinetics

An approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites is developed.

Genome Engineering With Zinc-Finger Nucleases

The history of ZFN development is reviewed, considerable progress has been made in methods for deriving zinc-finger sets for new genomic targets, but approaches to design and selection are still being perfected.

Enhancing the Specificity of Recombinase-Mediated Genome Engineering through Dimer Interface Redesign

The results provide a general means for improving hybrid recombinase specificity by protein engineering and illustrate the potential of these enzymes for basic research and therapeutic applications.



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

Using structure-based design, two variant ZFNs are engineer that 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.

Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

A structure-based approach to reducing off-target cleavage of zinc-finger nucleases by preventing homodimerization and lowering the dimerization energy is described, which increased the specificity of target site cleavage.

Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion

It is demonstrated that two autonomous ZFN pairs can be directed simultaneously to two different sites to induce a chromosomal deletion in ∼10% of alleles and will prove useful in targeted genome engineering approaches wherever an application requires the expression of two distinct Z FN pairs.

Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases.

Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.

The deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease and the zinc finger motifs opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications.

Expanding or restricting the target site repertoire of zinc-finger nucleases: the inter-domain linker as a major determinant of target site selectivity.

Both sequence and length of the inter-domain linker determine ZFN activity and target-site specificity, and are therefore important parameters to account for when designing ZFNs for genome editing.

Highly efficient endogenous human gene correction using designed zinc-finger nucleases

It is shown that zinc-finger nucleases designed against an X-linked severe combined immune deficiency mutation in the IL2Rγ gene yielded more than 18% gene-modified human cells without selection, raising the possibility of strategies based on zinc- finger nucleases for the treatment of disease.

The discovery of zinc fingers and their applications in gene regulation and genome manipulation.

  • A. Klug
  • Biology, Chemistry
    Annual review of biochemistry
  • 2010
The zinc finger design is ideally suited for engineering proteins to target specific genes, and several applications of such engineered zinc finger proteins are described here, including some of therapeutic importance.

Targeted transgene integration in plant cells using designed zinc finger nucleases

It is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci and point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.

Transient cold shock enhances zinc-finger nuclease–mediated gene disruption

Zinc-finger nucleases (ZFNs) are powerful tools for editing the genomes of cell lines and model organisms. Given the breadth of their potential application, simple methods that increase ZFN activity,