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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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revision of this article and B. Ewen-Campen, D. Yang-Zhou and N. Perrimon (Harvard Medical School, USA) for sharing the unpublished nub-Gal4 UAS–Cas9 stock.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.)).
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.
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Port, F., Bullock, S. Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nat Methods 13, 852–854 (2016). https://doi.org/10.1038/nmeth.3972
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