Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are hybrids between a nonspecific DNA-cleavage domain and a DNA-binding domain composed of Cys(2)His(2) zinc fingers. Because zinc fingers can be manipulated to recognize a broad range of sequences, these enzymes have the potential to direct cleavage to arbitrarily chose...

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Bibliographic Details
Published in:Genetics (Austin) 2002-07, Vol.161 (3), p.1169-1175
Main Authors: Bibikova, Marina, Golic, Mary, Golic, Kent G, Carroll, Dana
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Base Sequence
Crosses, Genetic
Deoxyribonucleases - genetics
Deoxyribonucleic acid
DNA
DNA Transposable Elements
Drosophila - genetics
Enzymes
Female
Genes
Genetic Vectors
Male
Mutagenesis, Insertional
Mutation
Ribonucleases - genetics
Transformation, Genetic
Zinc Fingers - genetics
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Summary:Zinc-finger nucleases (ZFNs) are hybrids between a nonspecific DNA-cleavage domain and a DNA-binding domain composed of Cys(2)His(2) zinc fingers. Because zinc fingers can be manipulated to recognize a broad range of sequences, these enzymes have the potential to direct cleavage to arbitrarily chosen targets. We have tested this idea by designing a pair of ZFNs that recognize a unique site in the yellow (y) gene of Drosophila. When these nucleases were expressed in developing larvae, they led to somatic mutations specifically in the y gene. These somatic mosaics were observed in approximately one-half of the males expressing both nucleases. Germline y mutations were recovered from 5.7% of males, but from none of the females, tested. DNA sequences were determined and showed that all of the mutations were small deletions and/or insertions located precisely at the designed target. These are exactly the types of alterations expected from nonhomologous end joining (NHEJ) following double-strand cleavage of the target. This approach promises to permit generation of directed mutations in many types of cells and organisms.
ISSN:0016-6731
1943-2631
1943-2631
DOI:10.1093/genetics/161.3.1169