Efficient Gene Targeting in Drosophila With Zinc-Finger Nucleases

Beumer, Kelly J.; Bhattacharyya, Gargi; Bibikova, Marina; Trautman, Jonathan K.; Carroll, Dana

doi:10.1534/genetics.105.052829

Cited by 224 publications

(229 citation statements)

References 62 publications

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“…In many cases, ZFN-mediated DSBs have been used to stimulate the HR DNA-repair machinery for HR-mediated gene replacement and gene addition. This approach has been successfully implemented in animals (Beumer et al, 2006;Meng et al, 2008), human cell lines (Urnov et al, 2005;Lombardo et al, 2007), and plants (Wright et al, 2005;Cai et al, 2009;Shukla et al, 2009;Townsend et al, 2009;de Pater et al, 2013). However, it is important to note that while ZFNinduced DSBs can indeed induce the HR repair machinery, most of these breaks are repaired by the cell's NHEJ DNA-repair machinery in plant and other species.…”

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confidence: 99%

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

2013

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Stimulation of the homologous recombination DNA-repair pathway via the induction of genomic double-strand breaks (DSBs) by zinc finger nucleases (ZFNs) has been deployed for gene replacement in plant cells. Nonhomologous end joining (NHEJ)-mediated repair of DSBs, on the other hand, has been utilized for the induction of site-specific mutagenesis in plants. Since NHEJ is the dominant DSB repair pathway and can also lead to the capture of foreign DNA molecules, we suggest that it can also be deployed for gene replacement. An acceptor DNA molecule in which a green fluorescent protein (GFP) coding sequence (gfp) was flanked by ZFN recognition sequences was used to produce transgenic target plants. A donor DNA molecule in which a promoterless hygromycin B phosphotransferase-encoding gene (hpt) was flanked by ZFN recognition sequences was constructed. The donor DNA molecule and ZFN expression cassette were delivered into target plants. ZFN-mediated sitespecific mutagenesis and complete removal of the GFP coding sequence resulted in the recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was unlinked to the acceptor DNA. More importantly, ZFNmediated digestion of both donor and acceptor DNA molecules resulted in NHEJ-mediated replacement of the gfp with hpt and recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was physically linked to the acceptor DNA. Sequence and phenotypical analyses, and transmission of the replacement events to the next generation, confirmed the stability of the NHEJ-induced gene exchange, suggesting its use as a novel method for transgene replacement and gene stacking in plants.

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Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

2013

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“…These custom zinc finger proteins can be attached to other protein domains to direct various functions to specific locations in the genomes of cells and organisms (10). When fused to the nonspecific DNA cleavage domain of the FokI restriction endonuclease, zinc fingers can direct double-strand breaks (DSBs) to disrupt specific genes (11)(12)(13)(14) or in combination with a repair template to induce gene correction via homologous recombination (HR) (15)(16)(17)(18). Thus, ZFNs show promise for the treatment of monogenic disorders by promoting the knockout or correction of specific genes.…”

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Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells

Mittelman

Moye²,

Morton

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Expanded triplet repeats have been identified as the genetic basis for a growing number of neurological and skeletal disorders. To examine the contribution of double-strand break repair to CAG⅐CTG repeat instability in mammalian systems, we developed zinc finger nucleases (ZFNs) that recognize and cleave CAG repeat sequences. Engineered ZFNs use a tandem array of zinc fingers, fused to the FokI DNA cleavage domain, to direct double-strand breaks (DSBs) in a site-specific manner. We first determined that the ZFNs cleave CAG repeats in vitro. Then, using our previously described tissue culture assay for identifying modifiers of CAG repeat instability, we found that transfection of ZFN-expression vectors induced up to a 15-fold increase in changes to the CAG repeat in human and rodent cell lines, and that longer repeats were much more sensitive to cleavage than shorter ones. Analysis of individual colonies arising after treatment revealed a spectrum of events consistent with ZFN-induced DSBs and dominated by repeat contractions. We also found that expressing a dominant-negative form of RAD51 in combination with a ZFN, dramatically reduced the effect of the nuclease, suggesting that DSB-induced repeat instability is mediated, in part, through homology directed repair. These studies identify a ZFN as a useful reagent for characterizing the effects of DSBs on CAG repeats in cells.DNA repair ͉ gene targeting ͉ triplet repeat instability ͉ zinc finger nucleases

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“…Meganucleases such as I-SceI are valuable cleavage reagents because of their long recognition sites (19-21), but their sites must be introduced before they can be attacked. Because zinc fingers exist for many of the DNA triplets, nucleases of this type can theoretically be created to target almost any preexisting locus.Studies performed in Drosophila and in human cells have shown that ZFNs can be designed to effectively cleave specific sequences (22)(23)(24)(25)(26)(27)(28). Additional studies with synthetic targets have demonstrated the efficiency of ZFN-directed cleavage in Xenopus, in mammalian cells and in plants (16,(29)(30)(31).…”

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Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells

Morton

Davis

Jørgensen

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Zinc-finger nucleases are chimeric proteins consisting of engineered zinc-finger DNA-binding motifs attached to an endonuclease domain. These proteins can induce site-specific DNA double-strand breaks in genomic DNA, which are then substrates for cellular repair mechanisms. Here, we demonstrate that engineered zinc-finger nucleases function effectively in somatic cells of the nematode Caenorhabditis elegans. Although gene-conversion events were indistinguishable from uncut DNA in our assay, nonhomologous end joining resulted in mutations at the target site. A synthetic target on an extrachromosomal array was targeted with a previously characterized nuclease, and an endogenous genomic sequence was targeted with a pair of specifically designed nucleases. In both cases, Ϸ20% of the target sites were mutated after induction of the corresponding nucleases. Alterations in the extrachromosomal targets were largely products of end-filling and blunt ligation. By contrast, alterations in the chromosomal target were mostly deletions. We interpret these differences to reflect the abundance of homologous templates present in the extrachromosomal arrays versus the paucity of such templates for repair of chromosomal breaks. In addition, we find evidence for the involvement of error-prone DNA synthesis in both homologous and nonhomologous pathways of repair. 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.DNA repair ͉ gene targeting ͉ nematodes ͉ nonhomologous end joining T he ability to make targeted double-strand breaks in chromosomal DNA has several important uses. It allows the detailed study of DNA repair mechanisms; it leads to localized mutagenesis at the break site; and it enhances the efficiency of targeted gene replacement through homologous recombination. We have been exploring the capabilities of one class of targetable cleavage reagents, the zinc-finger nucleases (ZFNs).ZFNs are chimeric proteins composed of DNA-binding Cys 2 His 2 zinc fingers fused to the nonspecific nuclease domain of the restriction enzyme FokI (1). Each finger makes contact primarily with a separate DNA triplet (2, 3). Natural and artificial zinc fingers have been characterized that bind to all 5Ј-GNN-3Ј, many ANN and CNN, and some TNN triplets (4-7). Furthermore, the modular nature of the zinc fingers allows them to be joined in essentially arbitrary combinations. Typically, three zinc fingers are combined to bind to a specific 9-bp DNA sequence with the nanomolar affinity required to be biologically useful, but additional fingers can be incorporated to confer increased specificity (8-11). Zinc-finger fusions to various functional domains have been used to create artificial transcription factors and DNA-modifying proteins (12,13).When attached to the FokI nuclease domain, zinc fingers can direct cleavage to specific DNA sequences. The nuclease domain must dimerize to cleave DNA (14), and because the di...

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Efficient Gene Targeting in Drosophila With Zinc-Finger Nucleases

Cited by 224 publications

References 62 publications

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells

Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells

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