Advertisement

CRISPR/Cas9 Genome Editing to Study Nervous System Development in Drosophila

  • Cornelia FritschEmail author
  • Simon G. Sprecher
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2047)

Abstract

Continuous implementation of new techniques allowing increasingly precise genetic manipulations makes the fruit fly Drosophila melanogaster an impacting model to study the nervous system. While transgenic approaches have been heavily used to investigate how the brain develops, genome editing has been notoriously hard in the fruit fly. The advent of versatile CRISPR/Cas9-based genome editing techniques allow the generation of engineered loci using homologous repair to replace the endogenous genome sequence with a designed template of interest. We here provide a protocol to generate an FRT/FLP-based conditional GFP or HA-flagged gene knockout.

Keywords

Drosophila Brain Genome editing CRISPR Conditional alleles 

Notes

Acknowledgments

We thank S. Bullock, M. Harrison, K. O’Connor-Giles, and J. Wildoger for plasmids. Special thanks to Jenifer Kaldun for generating the pBsF3xHAF plasmid. We thank the Bloomington Drosophila Stock center for fly strains. This work was funded by the Swiss National Science Foundation (31003A_149499 to S.G.S.).

References

  1. 1.
    Consortium IMK, Collins F, Rossant J, Wurst W (2007) A mouse for all reasons. Cell 128(1):9–13. . Epub 2007/01/16.  https://doi.org/10.1016/j.cell.2006.12.018CrossRefGoogle Scholar
  2. 2.
    Guan C, Ye C, Yang X, Gao J (2010) A review of current large-scale mouse knockout efforts. Genesis 48(2):73–85. . Epub 2010/01/23.  https://doi.org/10.1002/dvg.20594CrossRefPubMedGoogle Scholar
  3. 3.
    Jin M, Eblimit A, Pulikkathara M, Corr S, Chen R, Mardon G (2016) Conditional knockout of retinal determination genes in differentiating cells in Drosophila. FEBS J 283(15):2754–2766.  https://doi.org/10.1111/febs.13772. Epub 2016/06/04CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Xue Z, Ren M, Wu M, Dai J, Rong Y, Gao G (2014) Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila. G3 (Bethesda) 4(5):925–929.  https://doi.org/10.1534/g3.114.010496. Epub 2014/03/25CrossRefPubMedCentralGoogle Scholar
  5. 5.
    Schertel C, Albacara M, Rockel-Bauer C, Kelley N, Bischof J, Hens K et al (2015) A large-scale, in vivo transcription factor screen defines bivalent chromatin as a key property of regulatory factors mediating Drosophila wing development. Genome Res 25(4):514–523.  https://doi.org/10.1101/gr.181305.114. Epub 2015/01/09CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. Epub 2012/06/30.  https://doi.org/10.1126/science.1225829CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Bassett A, Tibbit C, Ponting C, Liu J (2013) Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep 4(1):220–228.  https://doi.org/10.1016/j.celrep.2013.06.020. Epub 2013/07/06CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Bier E, Harrison M, O’Connor-Giles K, Wildonger J (2018) Advances in engineering the fly genome with the CRISPR-Cas system. Genetics 208(1):1–18.  https://doi.org/10.1534/genetics.117.1113. Epub 2018/01/06CrossRefPubMedGoogle Scholar
  9. 9.
    Gratz S, Cummings A, Nguyen J, Hamm D, Donohue L, Harrison M et al (2013) Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194(4):1029–1035.  https://doi.org/10.1534/genetics.113.152710. Epub 2013/05/28CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Gratz S, Ukken F, Rubinstein C, Thiede G, Donohue L, Cummings A et al (2014) Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196(4):961–971.  https://doi.org/10.1534/genetics.113.160713. Epub 2014/01/31CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kondo S, Ueda R (2013) Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics 195(3):715–721.  https://doi.org/10.1534/genetics.113.156737. Epub 2013/09/05CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Korona D, Koestler S, Russell S (2017) Engineering the Drosophila genome for developmental biology. J Dev Biol 5(4).  https://doi.org/10.3390/jdb5040016. Epub 2018/04/05CrossRefGoogle Scholar
  13. 13.
    Port F, Chen H, Lee T, Bullock S (2014) Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A 111(29):E2967–E2976.  https://doi.org/10.1073/pnas.1405500111. Epub 2014/07/09CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Ren X, Sun J, Housden B, Hu Y, Roesel C, Lin S et al (2013) Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. Proc Natl Acad Sci U S A 110(47):19012–19017.  https://doi.org/10.1073/pnas.1318481110. Epub 2013/11/06CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Xu J, Ren X, Sun J, Wang X, Qiao H-H, Xu B-W et al (2015) A toolkit of CRISPR-based genome editing systems in Drosophila. J Genet Genomics 42(4):141–149.  https://doi.org/10.1016/j.jgg.2015.02.007CrossRefPubMedGoogle Scholar
  16. 16.
    Yu Z, Chen H, Liu J, Zhang H, Yan Y, Zhu N et al (2014) Various applications of TALEN- and CRISPR/Cas9-mediated homologous recombination to modify the Drosophila genome. Biol Open 3(4):271–280.  https://doi.org/10.1242/bio.20147682. Epub 2014/03/25CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Yu Z, Ren M, Wang Z, Zhang B, Rong Y, Jiao R et al (2013) Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila. Genetics 195(1):289–291.  https://doi.org/10.1534/genetics.113.153825. Epub 2013/07/09CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Elliott D, Brand A (2008) The GAL4 system : a versatile system for the expression of genes. Methods Mol Biol 420:79–95. Epub 2008/07/22.  https://doi.org/10.1007/978-1-59745-583-1_5CrossRefPubMedGoogle Scholar
  19. 19.
    Brand A, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118(2):401–415PubMedPubMedCentralGoogle Scholar
  20. 20.
    Golic K, Lindquist S (1989) The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59(3):499–509. Epub 1989/11/03. doi:.  https://doi.org/10.1016/0092-8674(89)90033-0CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Duffy J, Harrison D, Perrimon N (1998) Identifying loci required for follicular patterning using directed mosaics. Development 125(12):2263–2271PubMedGoogle Scholar
  22. 22.
    Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D (1994) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Science 263(5148):802–805CrossRefGoogle Scholar
  23. 23.
    Jennett A, Rubin G, Ngo T, Shepherd D, Murphy C, Dionne H et al (2012) A GAL4-driver line resource for Drosophila neurobiology. Cell Rep 2(4):991–1001.  https://doi.org/10.1016/j.celrep.2012.09.011CrossRefGoogle Scholar
  24. 24.
    Pfeiffer B, Jenett A, Hammonds A, Ngo T, Misra S, Murphy C et al (2008) Tools for neuroanatomy and neurogenetics in Drosophila. Proc Natl Acad Sci U S A 105(28):9715–9720.  https://doi.org/10.1073/pnas.0803697105. Epub 2008/07/16CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Gibson D, Young L, Chuang R-Y, Venter J, Hutchinson C, Smith H (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345. Epub 2009/04/14.  https://doi.org/10.1038/nmeth.1318CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of BiologyUniversity of FribourgFribourgSwitzerland

Personalised recommendations