RESUMEN
Neuronal tau reduction confers resilience against ß-amyloid and tau-related neurotoxicity in vitro and in vivo. Here, we introduce a novel translational approach to lower expression of the tau gene MAPT at the transcriptional level using gene-silencing zinc finger protein transcription factors (ZFP-TFs). Following a single administration of adeno-associated virus (AAV), either locally into the hippocampus or intravenously to enable whole-brain transduction, we selectively reduced tau messenger RNA and protein by 50 to 80% out to 11 months, the longest time point studied. Sustained tau lowering was achieved without detectable off-target effects, overt histopathological changes, or molecular alterations. Tau reduction with AAV ZFP-TFs was able to rescue neuronal damage around amyloid plaques in a mouse model of Alzheimer's disease (APP/PS1 line). The highly specific, durable, and controlled knockdown of endogenous tau makes AAV-delivered ZFP-TFs a promising approach for the treatment of tau-related human brain diseases.
Asunto(s)
Enfermedad de Alzheimer , Factores de Transcripción , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Ratones , Placa Amiloide/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de Zinc/genética , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.
Asunto(s)
Alelos , Proteína Huntingtina/genética , Enfermedad de Huntington/terapia , Mutación , Transcripción Genética , Dedos de Zinc , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Enfermedad de Huntington/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Neuroprotección , Repeticiones de TrinucleótidosRESUMEN
Modern agriculture demands crops carrying multiple traits. The current paradigm of randomly integrating and sorting independently segregating transgenes creates severe downstream breeding challenges. A versatile, generally applicable solution is hereby provided: the combination of high-efficiency targeted genome editing driven by engineered zinc finger nucleases (ZFNs) with modular 'trait landing pads' (TLPs) that allow 'mix-and-match', on-demand transgene integration and trait stacking in crop plants. We illustrate the utility of nuclease-driven TLP technology by applying it to the stacking of herbicide resistance traits. We first integrated into the maize genome an herbicide resistance gene, pat, flanked with a TLP (ZFN target sites and sequences homologous to incoming DNA) using WHISKERS™-mediated transformation of embryogenic suspension cultures. We established a method for targeted transgene integration based on microparticle bombardment of immature embryos and used it to deliver a second trait precisely into the TLP via cotransformation with a donor DNA containing a second herbicide resistance gene, aad1, flanked by sequences homologous to the integrated TLP along with a corresponding ZFN expression construct. Remarkably, up to 5% of the embryo-derived transgenic events integrated the aad1 transgene precisely at the TLP, that is, directly adjacent to the pat transgene. Importantly and consistent with the juxtaposition achieved via nuclease-driven TLP technology, both herbicide resistance traits cosegregated in subsequent generations, thereby demonstrating linkage of the two independently transformed transgenes. Because ZFN-mediated targeted transgene integration is becoming applicable across an increasing number of crop species, this work exemplifies a simple, facile and rapid approach to trait stacking.
Asunto(s)
Endonucleasas/genética , Marcación de Gen/métodos , Genoma de Planta/genética , Resistencia a los Herbicidas , Herbicidas/farmacología , Zea mays/genética , Productos Agrícolas , Endonucleasas/metabolismo , Ligamiento Genético , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Transgenes , Dedos de ZincRESUMEN
Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. To this end, designed transcriptional activators have been constructed by fusing transcriptional activation domains to DNA-binding proteins. In this study, a transcriptional activator from the herpes simplex virus, VP16, was used to identify plant regulatory proteins. Transcriptional activation domains were identified from each protein and fused with zinc finger DNA-binding proteins (ZFPs) to generate designed transcriptional activators. In addition, specific sequences within each transcriptional activation domain were modified to mimic the VP16 contact motif that interacts directly with RNA polymerase II core transcription factors. To evaluate these designed transcriptional activators, test systems were built in yeast and tobacco comprising reporter genes driven by promoters containing ZFP-binding sites upstream of the transcriptional start site. In yeast, transcriptional domains from the plant proteins ERF2 and PTI4 activated MEL1 reporter gene expression to levels similar to VP16 and the modified sequences displayed even greater levels of activation. Following stable transformation of the tobacco reporter system with transcriptional activators derived from ERF2, GUS reporter gene transcript accumulation was equal to or greater than those derived from VP16. Moreover, a modified ERF2 domain displayed significantly enhanced transcriptional activation compared with VP16 and with the unmodified ERF2 sequence. These results demonstrate that plant sequences capable of facilitating transcriptional activation can be found and, when fused to DNA-binding proteins, can enhance gene expression.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Nicotiana/genética , Ingeniería de Proteínas , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Cromosomas de las Plantas/genética , Evolución Molecular , Genes Reporteros , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/química , Estructura Terciaria de Proteína , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Transcripción GenéticaRESUMEN
Malaria afflicts over 200 million people worldwide, and its most lethal etiologic agent, Plasmodium falciparum, is evolving to resist even the latest-generation therapeutics. Efficient tools for genome-directed investigations of P. falciparum-induced pathogenesis, including drug-resistance mechanisms, are clearly required. Here we report rapid and targeted genetic engineering of this parasite using zinc-finger nucleases (ZFNs) that produce a double-strand break in a user-defined locus and trigger homology-directed repair. Targeting an integrated egfp locus, we obtained gene-deletion parasites with unprecedented speed (2 weeks), both with and without direct selection. ZFNs engineered against the parasite gene pfcrt, responsible for escape under chloroquine treatment, rapidly produced parasites that carried either an allelic replacement or a panel of specified point mutations. This method will enable a diverse array of genome-editing approaches to interrogate this human pathogen.
Asunto(s)
Endonucleasas/fisiología , Genoma de Protozoos , Plasmodium falciparum/genética , Ingeniería de Proteínas/métodos , Dedos de Zinc/fisiología , Alelos , Secuencia de Bases , Cloroquina/farmacología , Resistencia a Medicamentos/genética , Endonucleasas/genética , Datos de Secuencia Molecular , Plasmodium falciparum/efectos de los fármacos , Dedos de Zinc/genéticaRESUMEN
Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).
Asunto(s)
Células Madre Embrionarias/fisiología , Endonucleasas/metabolismo , Marcación de Gen/métodos , Ingeniería Genética/métodos , Células Madre Pluripotentes Inducidas/fisiología , Factores de Transcripción/metabolismo , Secuencia de Bases , Endonucleasas/genética , Proteínas de Homeodominio/genética , Humanos , Datos de Secuencia Molecular , Fosfatasa de Miosina de Cadena Ligera/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Factores de Transcripción/genética , Dedos de ZincRESUMEN
Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with â¼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.
Asunto(s)
Desoxirribonucleasas/química , Desoxirribonucleasas/genética , Inmunoglobulina M/genética , Ingeniería de Proteínas/métodos , Dedos de Zinc , Alelos , Animales , Secuencia de Bases , Exones/genética , Femenino , Técnicas de Inactivación de Genes , Sitios Genéticos/genética , Humanos , Inmunoglobulina G/genética , Inmunoglobulina M/deficiencia , Masculino , Microinyecciones , Datos de Secuencia Molecular , Mutación/genética , Oocitos/metabolismo , ARN Mensajero/genética , Conejos , Reproducibilidad de los ResultadosRESUMEN
Evolutionary studies necessary to dissect diverse biological processes have been limited by the lack of reverse genetic approaches in most organisms with sequenced genomes. We established a broadly applicable strategy using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for targeted disruption of endogenous genes and cis-acting regulatory elements in diverged nematode species.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Técnicas Genéticas , Genoma de los Helmintos , Elementos Reguladores de la Transcripción/genética , Dedos de Zinc , Animales , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Marcación de Gen , Genes de Helminto , Mutación INDEL , Mutagénesis , Factores de Transcripción/química , TransgenesRESUMEN
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.
Asunto(s)
Alelos , Proteínas Portadoras/genética , Desoxirribonucleasas/genética , Marcación de Gen/métodos , Proteínas de Xenopus/genética , Animales , Animales Modificados Genéticamente , Proteínas Portadoras/metabolismo , Desoxirribonucleasas/metabolismo , Xenopus , Proteínas de Xenopus/metabolismo , Dedos de ZincRESUMEN
Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.
Asunto(s)
Clatrina/metabolismo , Endocitosis , Animales , Secuencia de Bases , Linaje de la Célula , Membrana Celular/metabolismo , Dinamina II/metabolismo , Genoma , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Fluorescente/métodos , Modelos Genéticos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.
Asunto(s)
Desoxirribonucleasas/metabolismo , Células Madre Embrionarias/fisiología , Marcación de Gen/métodos , Células Madre Pluripotentes/fisiología , Dedos de Zinc/fisiología , Línea Celular , Desoxirribonucleasas/genética , Expresión Génica , Silenciador del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.
Asunto(s)
Endodesoxirribonucleasas/metabolismo , Técnicas de Inactivación de Genes , Inmunoglobulina M/genética , Microinyecciones , Dedos de Zinc , Proteínas de Unión al GTP rab/genética , Animales , Secuencia de Bases , ADN , Embrión de Mamíferos , Endodesoxirribonucleasas/genética , Estudios de Factibilidad , Femenino , Proteínas Fluorescentes Verdes , Masculino , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , ARN Mensajero , Ratas , Dedos de Zinc/genéticaRESUMEN
Nucleocytoplasmic transport occurs through gigantic proteinaceous channels called nuclear pore complexes (NPCs). Translocation through the NPC is exquisitely selective and is mediated by interactions between soluble transport carriers and insoluble NPC proteins that contain phenylalanine-glycine (FG) repeats. Although most FG nucleoporins (Nups) are organized symmetrically about the planar axis of the nuclear envelope, very few localize exclusively to one side of the NPC. We constructed Saccharomyces cerevisiae mutants with asymmetric FG repeats either deleted or swapped to generate NPCs with inverted FG asymmetry. The mutant Nups localize properly within the NPC and exhibit exchanged binding specificity for the export factor Xpo1. Surprisingly, we were unable to detect any defects in the Kap95, Kap121, Xpo1, or mRNA transport pathways in cells expressing the mutant FG Nups. These findings suggest that the biased distribution of FG repeats is not required for major nucleocytoplasmic trafficking events across the NPC.