Available Technology
Zinc-finger nucleases: determining and improving target sequence specificity
Technology:
In vitro assay
Markets Addressed
Zinc-finger nucleases (ZFNs) are enzymes engineered to recognize and cleave desired target DNA sequences. A ZFN monomer consists of a zinc-finger DNA-binding domain fused with a nonspecific FokI restriction endonuclease cleavage domain. As the FokI nuclease domain must dimerize to cleave DNA, ZFNs are designed in pairs to recognize two unique sequences flanking a spacer sequence of variable length and to cleave only when bound as a dimer. In addition to providing powerful research tools, ZFNs are in development as therapeutic gene therapy agents. Since imperfect specificity of engineered zinc-fingers domains has been linked to cellular toxicity, methods for determining and improving the specificities of ZFNs should be of considerable interest to companies developing therapeutic ZFNs.
Innovations and Advantages
Researchers in the laboratory of Professor David Liu have developed an in vitro selection method to broadly examine the DNA cleavage specificity of active 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. These results were further validated in cultured human cells.
The finding have implications for the design, testing, and application of candidate therapeutic ZFNs with increased specificity, and in particular suggest that ZFN specificity can be enhanced by designing ZFNs with decreased binding affinity, by lowering ZFN expression and by choosing target sites that differ by at least three base pairs from their closest sequence relatives in the genome.
In vitro selection for ZFN-mediated cleavage: Preselection library members are concatemers (represented by arrows) of identical ZFN target sites lacking 5′ phosphates (P). L, left half-site; R, right half-site, S, spacer; and L′, S′ and R′ are sequences complementary to L, S and R, respectively. ZFN cleavage reveals a 5′ phosphate, which is required for ligation of sequencing adapters (red and blue). The only sequences that can be amplified by PCR using primers complementary to the red and blue adapters are sequences that have been cleaved twice and have adapters on both ends. DNA cleaved at adjacent sites is purified via gel electrophoresis and sequenced. A computational screening step after sequencing ensures that the filled-in spacer sequences (S and S′) are complementary and therefore from the same molecule.
Additional Information
Publications:
Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection, Pattanayak,V; Ramirez, CL; Joung, JK; Liu, DR. Nature Methods, published online 7 August 2011; doi:10.1038/nmeth.1670.
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Inventor(s):
Guilinger, John Paul
Liu, David R.
Pattanayak, Vikram
Categories:
For further information, please contact:
Vivian Berlin, Director of Business Development
(617) 496-0474
Reference Harvard Case #4086