Cited by 860CRISPR and Genetic EngineeringDNA Repair MechanismsRNA regulation and disease

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

Bibikova, Marina, Golic, Mary, Golic, Kent G, Carroll, Dana·2002·Genetics

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Abstract

Zinc-finger nucleases (ZFNs) are hybrids between a nonspecific DNA-cleavage domain and a DNA-binding domain composed of Cys2His2 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.

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Authors: Bibikova, M., Golic, M., Golic, K. G., & Carroll, D.
Affiliations: University of Utah (US)
Corresponding author: Carroll, Dana
ORCID: Golic, Kent G
Carroll, Dana
Venue: Genetics
Publisher: Oxford University Press (OUP)
Published: 2002-07-01
Volume: 161
Issue: 3
Pages: 1169-1175
ISSN: 0016-6731 (ISSN-L)
1943-2631
DOI: 10.1093/genetics/161.3.1169
OpenAlex: W1906155566
Cited by: 860
References: 50
Citations by year: 2026: 12 · 2025: 30 · 2024: 44 · 2023: 40 · 2022: 50 · 2021: 46 · 2020: 42 · 2019: 41 · 2018: 49 · 2017: 50 · 2016: 70 · 2015: 64 · 2014: 65 · 2013: 47 · 2012: 45
FWCI: 3.36
Type: journal article
Language: en
Open access: closed
Licence: academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
Locations: Genetics (publishedVersion)
PubMed (publishedVersion)
Funders: National Institutes of Health (7r01gm060700-12)
National Institute of General Medical Sciences (R01GM60700, R01 GM58504, R01GM058504)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Keywords: zinc finger nucleases, targeted mutagenesis, Drosophila, yellow gene, germline mutations, non-homologous end joining, double-strand DNA cleavage, C2H2 zinc finger, directed mutagenesis
MeSH terms: Amino Acid Sequence, Animals, Base Sequence, Crosses, Genetic, Deoxyribonucleases, DNA Transposable Elements, Drosophila, Female, Genetic Vectors, Male, Ribonucleases, Transformation, Genetic, Mutagenesis, Insertional, Zinc Fingers

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

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