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Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs


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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Figure 1: Multiplexed Cas9 sgRNA expression in Drosophila with the tRNA–sgRNA expression system.
Figure 2: The UAS–tRNA–sgRNA system increases mutagenesis efficiency and tissue specificity of conditional CRISPR.
Figure 3: Flanking tRNAs can enhance Cpf1 genome editing in vivo.

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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 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revision of this article and B. Ewen-Campen, D. Yang-Zhou and N. Perrimon (Harvard Medical School, USA) for sharing the unpublished nub-Gal4 UASCas9 stock.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.)).

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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.

Corresponding authors

Correspondence to Fillip Port or Simon L Bullock.

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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.

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Supplementary Figures 1–5, Supplementary Tables 1 and 2, and Supplementary Protocol. (PDF 4438 kb)

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Port, F., Bullock, S. Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nat Methods 13, 852–854 (2016).

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