Skip to main content
PMC home page
Genetics logoLink to Genetics
. 2002 Jul;161(3):1169–1175. doi: 10.1093/genetics/161.3.1169

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

Marina Bibikova 1, Mary Golic 1, Kent G Golic 1, Dana Carroll 1
PMCID: PMC1462166  PMID: 12136019

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (162.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams M. D., Celniker S. E., Holt R. A., Evans C. A., Gocayne J. D., Amanatides P. G., Scherer S. E., Li P. W., Hoskins R. A., Galle R. F. The genome sequence of Drosophila melanogaster. Science. 2000 Mar 24;287(5461):2185–2195. doi: 10.1126/science.287.5461.2185. [DOI] [PubMed] [Google Scholar]
  2. Beall E. L., Rio D. C. Drosophila IRBP/Ku p70 corresponds to the mutagen-sensitive mus309 gene and is involved in P-element excision in vivo. Genes Dev. 1996 Apr 15;10(8):921–933. doi: 10.1101/gad.10.8.921. [DOI] [PubMed] [Google Scholar]
  3. Beerli R. R., Segal D. J., Dreier B., Barbas C. F., 3rd Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14628–14633. doi: 10.1073/pnas.95.25.14628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Capecchi M. R. Altering the genome by homologous recombination. Science. 1989 Jun 16;244(4910):1288–1292. doi: 10.1126/science.2660260. [DOI] [PubMed] [Google Scholar]
  5. Choo Y., Sánchez-García I., Klug A. In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence. Nature. 1994 Dec 15;372(6507):642–645. doi: 10.1038/372642a0. [DOI] [PubMed] [Google Scholar]
  6. Cole-Strauss A., Yoon K., Xiang Y., Byrne B. C., Rice M. C., Gryn J., Holloman W. K., Kmiec E. B. Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide. Science. 1996 Sep 6;273(5280):1386–1389. doi: 10.1126/science.273.5280.1386. [DOI] [PubMed] [Google Scholar]
  7. Dray T., Gloor G. B. Homology requirements for targeting heterologous sequences during P-induced gap repair in Drosophila melanogaster. Genetics. 1997 Oct;147(2):689–699. doi: 10.1093/genetics/147.2.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dreier B., Beerli R. R., Segal D. J., Flippin J. D., Barbas C. F., 3rd Development of zinc finger domains for recognition of the 5'-ANN-3' family of DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem. 2001 May 4;276(31):29466–29478. doi: 10.1074/jbc.M102604200. [DOI] [PubMed] [Google Scholar]
  9. Dreier B., Segal D. J., Barbas C. F., 3rd Insights into the molecular recognition of the 5'-GNN-3' family of DNA sequences by zinc finger domains. J Mol Biol. 2000 Nov 3;303(4):489–502. doi: 10.1006/jmbi.2000.4133. [DOI] [PubMed] [Google Scholar]
  10. Engels W. R., Johnson-Schlitz D. M., Eggleston W. B., Sved J. High-frequency P element loss in Drosophila is homolog dependent. Cell. 1990 Aug 10;62(3):515–525. doi: 10.1016/0092-8674(90)90016-8. [DOI] [PubMed] [Google Scholar]
  11. Engels W. R. Reversal of fortune for Drosophila geneticists? Science. 2000 Jun 16;288(5473):1973–1975. doi: 10.1126/science.288.5473.1973. [DOI] [PubMed] [Google Scholar]
  12. Geyer P. K., Corces V. G. Separate regulatory elements are responsible for the complex pattern of tissue-specific and developmental transcription of the yellow locus in Drosophila melanogaster. Genes Dev. 1987 Nov;1(9):996–1004. doi: 10.1101/gad.1.9.996. [DOI] [PubMed] [Google Scholar]
  13. Gloor G. B., Moretti J., Mouyal J., Keeler K. J. Distinct P-element excision products in somatic and germline cells of Drosophila melanogaster. Genetics. 2000 Aug;155(4):1821–1830. doi: 10.1093/genetics/155.4.1821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gloor G. B., Nassif N. A., Johnson-Schlitz D. M., Preston C. R., Engels W. R. Targeted gene replacement in Drosophila via P element-induced gap repair. Science. 1991 Sep 6;253(5024):1110–1117. doi: 10.1126/science.1653452. [DOI] [PubMed] [Google Scholar]
  15. Greisman H. A., Pabo C. O. A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites. Science. 1997 Jan 31;275(5300):657–661. doi: 10.1126/science.275.5300.657. [DOI] [PubMed] [Google Scholar]
  16. Havre P. A., Glazer P. M. Targeted mutagenesis of simian virus 40 DNA mediated by a triple helix-forming oligonucleotide. J Virol. 1993 Dec;67(12):7324–7331. doi: 10.1128/jvi.67.12.7324-7331.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Isalan M., Choo Y., Klug A. Synergy between adjacent zinc fingers in sequence-specific DNA recognition. Proc Natl Acad Sci U S A. 1997 May 27;94(11):5617–5621. doi: 10.1073/pnas.94.11.5617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Isalan M., Klug A., Choo Y. A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nat Biotechnol. 2001 Jul;19(7):656–660. doi: 10.1038/90264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Isalan M., Klug A., Choo Y. Comprehensive DNA recognition through concerted interactions from adjacent zinc fingers. Biochemistry. 1998 Sep 1;37(35):12026–12033. doi: 10.1021/bi981358z. [DOI] [PubMed] [Google Scholar]
  20. Jasin M. Genetic manipulation of genomes with rare-cutting endonucleases. Trends Genet. 1996 Jun;12(6):224–228. doi: 10.1016/0168-9525(96)10019-6. [DOI] [PubMed] [Google Scholar]
  21. Jeggo P. A. DNA breakage and repair. Adv Genet. 1998;38:185–218. doi: 10.1016/s0065-2660(08)60144-3. [DOI] [PubMed] [Google Scholar]
  22. Keeler K. J., Dray T., Penney J. E., Gloor G. B. Gene targeting of a plasmid-borne sequence to a double-strand DNA break in Drosophila melanogaster. Mol Cell Biol. 1996 Feb;16(2):522–528. doi: 10.1128/mcb.16.2.522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kim J. S., Pabo C. O. Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants. Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):2812–2817. doi: 10.1073/pnas.95.6.2812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kim Y. G., Cha J., Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1156–1160. doi: 10.1073/pnas.93.3.1156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Koller B. H., Smithies O. Altering genes in animals by gene targeting. Annu Rev Immunol. 1992;10:705–730. doi: 10.1146/annurev.iy.10.040192.003421. [DOI] [PubMed] [Google Scholar]
  26. Liu P. Q., Rebar E. J., Zhang L., Liu Q., Jamieson A. C., Liang Y., Qi H., Li P. X., Chen B., Mendel M. C. Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A. J Biol Chem. 2001 Jan 5;276(14):11323–11334. doi: 10.1074/jbc.M011172200. [DOI] [PubMed] [Google Scholar]
  27. Liu Q., Segal D. J., Ghiara J. B., Barbas C. F., 3rd Design of polydactyl zinc-finger proteins for unique addressing within complex genomes. Proc Natl Acad Sci U S A. 1997 May 27;94(11):5525–5530. doi: 10.1073/pnas.94.11.5525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mismer D., Rubin G. M. Analysis of the promoter of the ninaE opsin gene in Drosophila melanogaster. Genetics. 1987 Aug;116(4):565–578. doi: 10.1093/genetics/116.4.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moore M., Choo Y., Klug A. Design of polyzinc finger peptides with structured linkers. Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1432–1436. doi: 10.1073/pnas.98.4.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pabo C. O., Peisach E., Grant R. A. Design and selection of novel Cys2His2 zinc finger proteins. Annu Rev Biochem. 2001;70:313–340. doi: 10.1146/annurev.biochem.70.1.313. [DOI] [PubMed] [Google Scholar]
  31. Preston C. R., Sved J. A., Engels W. R. Flanking duplications and deletions associated with P-induced male recombination in Drosophila. Genetics. 1996 Dec;144(4):1623–1638. doi: 10.1093/genetics/144.4.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rong Y. S., Golic K. G. A targeted gene knockout in Drosophila. Genetics. 2001 Mar;157(3):1307–1312. doi: 10.1093/genetics/157.3.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rong Y. S., Golic K. G. Gene targeting by homologous recombination in Drosophila. Science. 2000 Jun 16;288(5473):2013–2018. doi: 10.1126/science.288.5473.2013. [DOI] [PubMed] [Google Scholar]
  34. Segal D. J., Dreier B., Beerli R. R., Barbas C. F., 3rd Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2758–2763. doi: 10.1073/pnas.96.6.2758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Smith J., Bibikova M., Whitby F. G., Reddy A. R., Chandrasegaran S., Carroll D. Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res. 2000 Sep 1;28(17):3361–3369. doi: 10.1093/nar/28.17.3361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Staveley B. E., Heslip T. R., Hodgetts R. B., Bell J. B. Protected P-element termini suggest a role for inverted-repeat-binding protein in transposase-induced gap repair in Drosophila melanogaster. Genetics. 1995 Mar;139(3):1321–1329. doi: 10.1093/genetics/139.3.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Takasu-Ishikawa E., Yoshihara M., Hotta Y. Extra sequences found at P element excision sites in Drosophila melanogaster. Mol Gen Genet. 1992 Mar;232(1):17–23. doi: 10.1007/BF00299132. [DOI] [PubMed] [Google Scholar]
  38. Vasquez K. M., Marburger K., Intody Z., Wilson J. H. Manipulating the mammalian genome by homologous recombination. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8403–8410. doi: 10.1073/pnas.111009698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wang G., Levy D. D., Seidman M. M., Glazer P. M. Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation. Mol Cell Biol. 1995 Mar;15(3):1759–1768. doi: 10.1128/mcb.15.3.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wang G., Seidman M. M., Glazer P. M. Mutagenesis in mammalian cells induced by triple helix formation and transcription-coupled repair. Science. 1996 Feb 9;271(5250):802–805. doi: 10.1126/science.271.5250.802. [DOI] [PubMed] [Google Scholar]
  41. Xu L., Zerby D., Huang Y., Ji H., Nyanguile O. F., de los Angeles J. E., Kadan M. J. A versatile framework for the design of ligand-dependent, transgene-specific transcription factors. Mol Ther. 2001 Feb;3(2):262–273. doi: 10.1006/mthe.2000.0254. [DOI] [PubMed] [Google Scholar]
  42. Zhang L., Spratt S. K., Liu Q., Johnstone B., Qi H., Raschke E. E., Jamieson A. C., Rebar E. J., Wolffe A. P., Case C. C. Synthetic zinc finger transcription factor action at an endogenous chromosomal site. Activation of the human erythropoietin gene. J Biol Chem. 2000 Oct 27;275(43):33850–33860. doi: 10.1074/jbc.M005341200. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES