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Precise Genome Editing of Drosophila with CRISPR RNA-Guided Cas9

  • Protocol
CRISPR

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1311))

Abstract

The readily programmable CRISPR-Cas9 system is transforming genome engineering. We and others have adapted the S. pyogenes CRISPR-Cas9 system to precisely engineer the Drosophila genome and demonstrated that these modifications are efficiently transmitted through the germline. Here we provide a detailed protocol for engineering small indels, defined deletions, and targeted insertion of exogenous DNA sequences within one month using a rapid DNA injection-based approach.

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Abbreviations

CRISPR:

Clustered regularly interspaced short palindromic repeats

crRNA:

CRISPR RNA

DSB:

Double-strand break

dsDNA:

Double-stranded DNA

gRNA:

Guide RNA

HDR:

Homology-directed repair

Indel:

Insertion-deletion

NHEJ:

Nonhomologous end joining

PAM:

Protospacer adjacent motif

ssDNA:

Single-stranded DNA

tracrRNA:

Trans-activating CRISPR RNA

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Acknowledgements

We are grateful to members of the Harrison, O’Connor-Giles, and Wildonger labs for their help in establishing CRISPR-Cas9 protocols in Drosophila, and to Dustin Rubinstein for comments on this chapter. Our work has been funded by start-up funds from the University of Wisconsin to M.M.H., J.W., and K.O.C.G. and grants from the National Institutes of Health to J.W. (R00 NS072252) and K.O.C.G. (R00 NS060985 and R01 NS078179). Plasmids and transgenic fly lines described here are available through the nonprofit distributor Addgene and the Bloomington Drosophila Stock Center, respectively. Detailed reagent information is available at http://flycrispr.molbio.wisc.edu.

Author information

Authors and Affiliations

  1. Genetics Training Program, University of Wisconsin-Madison, Madison, WI, 53706, USA

    Scott J. Gratz & Kate M. O’Connor-Giles

  2. Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53706, USA

    Melissa M. Harrison

  3. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA

    Jill Wildonger

  4. Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI, 53706, USA

    Kate M. O’Connor-Giles

  5. Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, 227D Robert M. Bock Labs, 1525 Linden Dr., Madison, WI, 53706, USA

    Kate M. O’Connor-Giles

Authors
  1. Scott J. Gratz
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  2. Melissa M. Harrison
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  3. Jill Wildonger
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  4. Kate M. O’Connor-Giles
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Corresponding author

Correspondence to Kate M. O’Connor-Giles .

Editor information

Editors and Affiliations

  1. Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden

    Magnus Lundgren

  2. Department of Molecular Biology MIMS, UmeĂĄ University, UmeĂĄ, Sweden

    Emmanuelle Charpentier

  3. Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand

    Peter C. Fineran

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Gratz, S.J., Harrison, M.M., Wildonger, J., O’Connor-Giles, K.M. (2015). Precise Genome Editing of Drosophila with CRISPR RNA-Guided Cas9. In: Lundgren, M., Charpentier, E., Fineran, P. (eds) CRISPR. Methods in Molecular Biology, vol 1311. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2687-9_22

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  • DOI: https://doi.org/10.1007/978-1-4939-2687-9_22

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2686-2

  • Online ISBN: 978-1-4939-2687-9

  • eBook Packages: Springer Protocols

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