Creating zinc finger nucleases to manipulate the genome in a site-specific manner using a modular-assembly approach.
Homologous recombination is the most precise way to manipulate the genome. It has been used extensively in bacteria, yeast, murine embryonic stem cells, and a few other specialized cell lines, but it has not been available in other genetic systems such as mammalian somatic cells. However, the creation of a gene-specific DNA double-strand break can stimulate homologous recombination by several-thousandfold in mammalian somatic cells. These double-strand breaks can be created in mammalian genomes by zinc finger nucleases (ZFNs), artificial proteins in which a zinc finger DNA-binding domain is fused to a nonspecific nuclease domain. This article describes how to identify potential targets for ZFN cutting. It also focuses on how to assemble ZFNs to recognize target sequences of the form 5'-GNNGNNGNN-3'. It is likely that there will be improvements in making the individual finger modules. High-throughput methods that combine both selection and assembly are being developed, which will broaden the number of sites that can be targeted, optimize the overall structure of the ZFNs, and increase the possible experimental and therapeutic uses for ZFNs.
Matthew
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Porteus
D004247:DNA / Q000235:genetics / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004247:DNA; D003851:Deoxyribonucleases