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1.
Nature ; 500(7462): 296-300, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23863942

RESUMO

Down's syndrome is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases, we inserted a large, inducible XIST transgene into the DYRK1A locus on chromosome 21, in Down's syndrome pluripotent stem cells. The XIST non-coding RNA coats chromosome 21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing and DNA methylation to form a 'chromosome 21 Barr body'. This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21, free from genetic and epigenetic noise. Notably, deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one chromosome 21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of 'chromosome therapy'.


Assuntos
Cromossomos Humanos Par 21/genética , Mecanismo Genético de Compensação de Dose , Síndrome de Down/genética , RNA Longo não Codificante/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Metilação de DNA , Síndrome de Down/terapia , Inativação Gênica , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Camundongos , Mutagênese Insercional , Neurogênese , RNA Longo não Codificante/genética , Cromatina Sexual/genética , Inativação do Cromossomo X/genética
2.
Nat Methods ; 12(5): 465-71, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25799440

RESUMO

Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.


Assuntos
Regulação da Expressão Gênica/fisiologia , Genoma , Edição de RNA/fisiologia , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , DNA/genética , Ensaio de Imunoadsorção Enzimática , Marcadores Genéticos , Fatores de Transcrição/genética
3.
Proc Natl Acad Sci U S A ; 108(17): 7052-7, 2011 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-21471457

RESUMO

The frog Xenopus, an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. Here, we address this problem by genome editing with zinc-finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in Xenopus tropicalis induced mutations consistent with nonhomologous end joining at the target site, resulting in mosaic loss of the fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles, and founder animals yielded progeny carrying insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity between three germ-line mutant alleles. Because ZFNs can be designed against any locus, our data provide a generally applicable protocol for gene disruption in Xenopus.


Assuntos
Alelos , Proteínas de Transporte/genética , Desoxirribonucleases/genética , Marcação de Genes/métodos , Proteínas de Xenopus/genética , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/metabolismo , Desoxirribonucleases/metabolismo , Xenopus , Proteínas de Xenopus/metabolismo , Dedos de Zinco
4.
Biotechnol Bioeng ; 106(1): 97-105, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20047187

RESUMO

Mammalian cells with multi-gene knockouts could be of considerable utility in research, drug discovery, and cell-based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events--a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc-finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi-locus genome engineering.


Assuntos
Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Técnicas de Inativação de Genes/métodos , Dedos de Zinco , Animais , Células CHO , Cricetinae , Cricetulus
5.
Nat Biotechnol ; 25(7): 778-85, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17603475

RESUMO

Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs 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. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.


Assuntos
Biotecnologia/métodos , Dedos de Zinco , Sequência de Bases , Sítios de Ligação , Catálise , Desoxirribonucleases de Sítio Específico do Tipo II/química , Dimerização , Genoma , Proteínas de Fluorescência Verde/química , Humanos , Células K562 , Modelos Biológicos , Conformação Molecular , Dados de Sequência Molecular , Estrutura Terciária de Proteína
6.
Stem Cell Reports ; 4(4): 569-77, 2015 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-25772471

RESUMO

Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources-potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Marcação de Genes , Células-Tronco Pluripotentes Induzidas/metabolismo , Alelos , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Endonucleases/genética , Endonucleases/metabolismo , Expressão Gênica , Marcação de Genes/métodos , Vetores Genéticos/genética , Genótipo , Recombinação Homóloga , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mutação , Reparo de DNA por Recombinação , Análise de Sequência de DNA , Dedos de Zinco/genética
7.
Methods Mol Biol ; 649: 247-56, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20680839

RESUMO

The development of zinc finger nucleases for targeted gene modification can benefit from rapid functional assays that directly quantify activity at the endogenous target. Here we describe a simple procedure for quantifying mutations that result from DNA double-strand break repair via non-homologous end joining. The assay is based on the ability of the Surveyor nuclease to selectively cleave distorted duplex DNA formed via cross-annealing of mutated and wild-type sequence.


Assuntos
Bioensaio/métodos , Animais , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Eletroforese em Gel de Poliacrilamida , Endonucleases/genética , Endonucleases/metabolismo , Humanos , Modelos Biológicos , Reação em Cadeia da Polimerase , Dedos de Zinco/genética
8.
Nat Biotechnol ; 26(7): 808-16, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18587387

RESUMO

Homozygosity for the naturally occurring Delta32 deletion in the HIV co-receptor CCR5 confers resistance to HIV-1 infection. We generated an HIV-resistant genotype de novo using engineered zinc-finger nucleases (ZFNs) to disrupt endogenous CCR5. Transient expression of CCR5 ZFNs permanently and specifically disrupted approximately 50% of CCR5 alleles in a pool of primary human CD4(+) T cells. Genetic disruption of CCR5 provided robust, stable and heritable protection against HIV-1 infection in vitro and in vivo in a NOG model of HIV infection. HIV-1-infected mice engrafted with ZFN-modified CD4(+) T cells had lower viral loads and higher CD4(+) T-cell counts than mice engrafted with wild-type CD4(+) T cells, consistent with the potential to reconstitute immune function in individuals with HIV/AIDS by maintenance of an HIV-resistant CD4(+) T-cell population. Thus adoptive transfer of ex vivo expanded CCR5 ZFN-modified autologous CD4(+) T cells in HIV patients is an attractive approach for the treatment of HIV-1 infection.


Assuntos
Transferência Adotiva/métodos , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/transplante , Desoxirribonucleases/genética , Infecções por HIV/prevenção & controle , Infecções por HIV/cirurgia , Dedos de Zinco/genética , Animais , Células Cultivadas , Mapeamento Cromossômico/métodos , Engenharia Genética/métodos , Humanos , Imunidade Inata , Camundongos , Resultado do Tratamento
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