Dana Carroll

..This article reviews discoveries that led to the development of ZFNs, cites examples of successes in genome engineering, and projects how ZFNs may be used in the future, particularly in applications to humans...

..The bright prospects for future applications of ZFNs, including human gene therapy, are discussed...

..New tools are available for the design and testing of ZFNs for new target genes. Applications of ZFNs to stem cells have been described, and genuine gene therapy trials appear to be on the immediate horizon...

..If all goes smoothly, design, construction and cloning can be completed in about two weeks, with expression and testing completed in one additional week...

..The injection procedure is much more rapid than earlier approaches and makes possible the generation and recovery of targeted gene alterations at essentially any locus within 2 fly generations...

..They also demonstrate that the outcome can be biased toward gene replacement by disabling the major NHEJ pathway and toward simple mutagenesis by interfering with the major HR process...

..We describe the construction of ZFNs for novel targets and their application to targeted mutagenesis and targeted gene replacement in Drosophila melanogaster and Caenorhabditis elegans...

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..A donor DNA carrying desired sequence changes can be incorporated in the injection mix and leads to targeted gene replacement, with particularly good efficiency when the recipient embryos are defective for nonhomologous end joining...

..These data indicate that ZFNs can form the basis of a highly efficient method for targeted mutagenesis of plant genes...

..Zinc-finger nucleases have the potential of being targetable to arbitrarily selected sites, owing to the flexibility of zinc finger recognition of DNA...

..DNA ligase IV is required for efficient end joining, particularly of blunt ends. In its absence, a secondary end-joining pathway relies more heavily on microhomologies in producing deletions...

..This approach promises to permit generation of directed mutations in many types of cells and organisms...

..Future work will be needed to translate these in vitro findings to in vivo applications and to determine whether zinc finger nucleases create undesired genomic instability...

..They will provide information on the best methods for creating models of human disease in experimental organisms and for modifying genes in applications to human gene therapy. ..

..Gene targeting can be used to create animal models for human disease and, ultimately, for human gene therapy. ..

..To demonstrate the generality of this approach to gene targeting, it will be applied to another mouse locus, the Rpo2-1 gene. ..

..To test the generality of the method, it will be applied to a second Drosophila locus, the rosy (ry) gene. ..

..This will prepare the way for targeting of specific sites in complex genomes. ..

..Gene targeting can be used to create animal models for human disease and, ultimately, for human gene therapy. ..