Targeted Chromosomal Cleavage and Mutagenesis in Drosophila Using Zinc-Finger Nucleases

Targeted Chromosomal Cleavage and Mutagenesis in Drosophila Using Zinc-Finger Nucleases Marina Bibikova a , Mary Golic b , Kent G. Golic b , and Dana Carroll a a Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132 b Department of Biology, University of Utah, Salt Lake City, Utah 84112 Corresponding author: Dana Carroll, University of Utah School of Medicine, Medical Research and Education Bldg., 20 North 1900 East, Salt Lake City, UT 84132., dana@biochem.utah.edu (E-mail) Communicating editor: S. H ENIKOFF 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genetics Genetics Society of America

Targeted Chromosomal Cleavage and Mutagenesis in Drosophila Using Zinc-Finger Nucleases

Genetics, Volume 161 (3): 1169 – Jul 1, 2002

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Publisher
Genetics Society of America
Copyright
Copyright © 2002 by the Genetics Society of America
ISSN
0016-6731
eISSN
1943-2631
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Abstract

Marina Bibikova a , Mary Golic b , Kent G. Golic b , and Dana Carroll a a Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132 b Department of Biology, University of Utah, Salt Lake City, Utah 84112 Corresponding author: Dana Carroll, University of Utah School of Medicine, Medical Research and Education Bldg., 20 North 1900 East, Salt Lake City, UT 84132., dana@biochem.utah.edu (E-mail) Communicating editor: S. H ENIKOFF 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.

Journal

GeneticsGenetics Society of America

Published: Jul 1, 2002

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