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  • 2010-2014  (2)
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  • 1
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    Comparing Zinc Finger Nucleases and Transcription Activator-Like Effector Nucleases for Gene Targeting in Drosophila (2013)
    Genetics Society of America (GSA)
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    Publication Date: 2013-10-09
    Description: Zinc-finger nucleases have proven to be successful as reagents for targeted genome manipulation in Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially greater than for zinc-finger nucleases , and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.
    Electronic ISSN: 2160-1836
    Topics: Biology
    Published by Genetics Society of America (GSA)
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  • 2
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    Donor DNA Utilization During Gene Targeting with Zinc-Finger Nucleases (2013)
    Genetics Society of America (GSA)
    Details
    Publication Date: 2013-04-10
    Description: Gene targeting is the term commonly applied to experimental gene replacement by homologous recombination (HR). This process is substantially stimulated by a double-strand break (DSB) in the genomic target. Zinc-finger nucleases (ZFNs) are targetable cleavage reagents that provide an effective means of introducing such a break in conjunction with delivery of a homologous donor DNA. In this study we explored several parameters of donor DNA structure during ZFN-mediated gene targeting in Drosophila melanogaster embryos, as follows. 1) We confirmed that HR outcomes are enhanced relative to the alternative nonhomologous end joining (NHEJ) repair pathway in flies lacking DNA ligase IV. 2) The minimum amount of homology needed to support efficient HR in fly embryos is between 200 and 500 bp. 3) Conversion tracts are very broad in this system: donor sequences more than 3 kb from the ZFN-induced break are found in the HR products at approximately 50% of the frequency of a marker at the break. 4) Deletions carried by the donor DNA are readily incorporated at the target. 5) While linear double-stranded DNAs are not effective as donors, single-stranded oligonucleotides are. These observations should enable better experimental design for gene targeting in Drosophila and help guide similar efforts in other systems.
    Electronic ISSN: 2160-1836
    Topics: Biology
    Published by Genetics Society of America (GSA)
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