RESUMO
An efficient method for making directed DNA sequence modifications to plant genes (gene targeting) is at present lacking, thereby frustrating efforts to dissect plant gene function and engineer crop plants that better meet the world's burgeoning need for food, fibre and fuel. Zinc-finger nucleases (ZFNs)-enzymes engineered to create DNA double-strand breaks at specific loci-are potent stimulators of gene targeting; for example, they can be used to precisely modify engineered reporter genes in plants. Here we demonstrate high-frequency ZFN-stimulated gene targeting at endogenous plant genes, namely the tobacco acetolactate synthase genes (ALS SuRA and SuRB), for which specific mutations are known to confer resistance to imidazolinone and sulphonylurea herbicides. Herbicide-resistance mutations were introduced into SuR loci by ZFN-mediated gene targeting at frequencies exceeding 2% of transformed cells for mutations as far as 1.3 kilobases from the ZFN cleavage site. More than 40% of recombinant plants had modifications in multiple SuR alleles. The observed high frequency of gene targeting indicates that it is now possible to efficiently make targeted sequence changes in endogenous plant genes.
Assuntos
Desoxirribonucleases/metabolismo , Marcação de Genes/métodos , Genes de Plantas/genética , Nicotiana/genética , Engenharia de Proteínas , Dedos de Zinco , Acetolactato Sintase/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Desoxirribonucleases/química , Desoxirribonucleases/genética , Alimentos Geneticamente Modificados , Resistência a Herbicidas/genética , Herbicidas/farmacologia , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Recombinação Genética/genética , Nicotiana/efeitos dos fármacos , Nicotiana/enzimologia , Transformação GenéticaRESUMO
Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an "open-source" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.
Assuntos
Endonucleases/metabolismo , Engenharia Genética/métodos , Dedos de Zinco , Sequência de Bases , Endonucleases/toxicidade , Marcação de Genes , Proteínas de Fluorescência Verde/genética , Humanos , Células K562 , Dados de Sequência Molecular , Mutagênese , Mutação/genética , Conformação ProteicaRESUMO
Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.