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Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs

Nature Methods volume 13, pages 852854 (2016) | Download Citation

Abstract

We present tRNA-based vectors for producing multiple clustered regularly interspaced short palindromic repeats (CRISPR) single guide RNAs (sgRNAs) from a single RNA polymerase II or III transcript in Drosophila. The system, which is based on liberation of sgRNAs by processing flanking tRNAs, permits highly efficient multiplexing of Cas9-based mutagenesis. We also demonstrate that the tRNA–sgRNA system markedly increases the efficacy of conditional gene disruption by Cas9 and can promote editing by the recently discovered RNA-guided endonuclease Cpf1.

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Acknowledgements

We thank N. Muschalik for extensive input and discussions, as well as other members of the Bullock lab and users of http://www.crisprflydesign.org for feedback. We are also grateful to M. Boutros (DKFZ, Germany) for support during revision of this article and B. Ewen-Campen, D. Yang-Zhou and N. Perrimon (Harvard Medical School, USA) for sharing the unpublished nub-Gal4 UASCas9 stock. This study was supported by a Marie-Curie IntraEuropean Fellowship (to F.P.) and core funding from the MRC (file reference number MC_U105178790 (to S.L.B.)).

Author information

Author notes

    • Fillip Port

    Present address: Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Affiliations

  1. Cell Biology Division, MRC Laboratory of Molecular Biology, Cambridge, UK.

    • Fillip Port
    •  & Simon L Bullock

Authors

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Contributions

F.P. conceived the study, designed experiments, performed experiments, analyzed data and wrote the manuscript. S.L.B. designed experiments, analyzed data and wrote the manuscript.

Competing interests

F.P. and S.L.B. are inventors of Cas9-expressing fly strains that have been licensed by the MRC to commercial providers of Drosophila injection services.

Corresponding authors

Correspondence to Fillip Port or Simon L Bullock.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–5, Supplementary Tables 1 and 2, and Supplementary Protocol.

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DOI

https://doi.org/10.1038/nmeth.3972

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