Designer zinc-finger proteins and their applications

Papworth, Monika; Kolasinska, Paulina; Minczuk, Michal

doi:10.1016/j.gene.2005.09.011

Cited by 131 publications

(99 citation statements)

References 120 publications

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“…Zinc fingers are predominantly DNA binding proteins adapted to operate in the nucleus. Even in the absence of nuclear localization signals they often localize in the nucleus (10). To use designer ZFPs to manipulate mtDNA, they have to be both effectively targeted to mitochondria and at the same time excluded from the nucleus to avoid binding to nuclear DNA, which could be toxic (12).…”

Section: Resultsmentioning

confidence: 99%

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Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase

Minczuk

Papworth

Kolasinska

et al. 2006

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

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143

112

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We used engineered zinc finger peptides (ZFPs) to bind selectively to predetermined sequences in human mtDNA. Surprisingly, we found that engineered ZFPs cannot be reliably routed to mitochondria by using only conventional mitochondrial targeting sequences. We here show that addition of a nuclear export signal allows zinc finger chimeric enzymes to be imported into human mitochondria. The selective binding of mitochondria-specific ZFPs to mtDNA was exemplified by targeting the T8993G mutation, which causes two mitochondrial diseases, neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) and also maternally inherited Leigh's syndrome. To develop a system that allows the monitoring of site-specific alteration of mtDNA we combined a ZFP with the easily assayed DNA-modifying activity of hDNMT3a methylase. Expression of the mutation-specific chimeric methylase resulted in the selective methylation of cytosines adjacent to the mutation site. This is a proof of principle that it is possible to target and alter mtDNA in a sequence-specific manner by using zinc finger technology.gene therapy ͉ mitochondria ͉ mitochondrial diseases ͉ synthetic zinc finger peptides ͉ nuclear export signal

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Section: Resultsmentioning

confidence: 99%

“…These have been used as powerful tools for intervening in nuclear gene expression and modifying DNA in a sequence-specific manner (6)(7)(8)(9)(10)(11). In all of the previous approaches the engineered zinc finger peptides (ZFPs) have been expressed from exogenous DNA templates, synthesized in the cytoplasm, and imported to the nucleus.…”

mentioning

confidence: 99%

Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase

Minczuk

Papworth

Kolasinska

et al. 2006

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

Self Cite

143

112

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“…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 dimer interface is weak (15), two nuclease domains are typically brought into close proximity by pairs of zinc-finger sets binding to neighboring sites.…”

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confidence: 99%

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confidence: 99%

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.

169

124

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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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“…To this end, we and others have employed engineered zinc-finger protein transcription factors (ZFP TFs), which employ natural transcriptional control mechanisms, to bring about the up-or downregulation of a variety of endogenous genes (reviewed in references [1][2][3][4][5][6] ). These factors can function with single gene specificity, 7,8 modulate stem cell fate, 9 and have shown promise in multiple pre-clinical animal studies, including models of angiogenesis [10][11][12][13] and experimental diabetic neuropathy, 14 as well as in multiple clinical trials.…”

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confidence: 99%

Regulation of endogenous gene expression using small molecule-controlled engineered zinc-finger protein transcription factors

et al. 2007

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Designer zinc-finger proteins and their applications

Cited by 131 publications

References 120 publications

Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase

Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase

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

Regulation of endogenous gene expression using small molecule-controlled engineered zinc-finger protein transcription factors

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