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 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.
Department of Biochemistry, University of Utah School of Medicine, Medical Research and Education Building, 20 North 1900 East, Salt Lake City, UT 84132, USA.
Marina
M
Bibikova
Mary
M
Golic
Kent G
KG
Golic
Dana
D
Carroll
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003433:Crosses, Genetic; D004251:DNA Transposable Elements; D003851:Deoxyribonucleases / Q000235:genetics*; D004330:Drosophila / Q000235:genetics*; D005260:Female; D005822:Genetic Vectors; D008297:Male; D016254:Mutagenesis, Insertional; D012260:Ribonucleases / Q000235:genetics*; D014170:Transformation, Genetic; D016335:Zinc Fingers / Q000235:genetics*
D004251:DNA Transposable Elements; D003851:Deoxyribonucleases; D012260:Ribonucleases