Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. Doudna, J.A. & Charpentier, E. Science 346, 1258096 (2014).

    Article  Google Scholar 

  2. Perez-Pinera, P. et al. Nat. Methods 10, 973–976 (2013).

    CAS  Article  Google Scholar 

  3. Mali, P. et al. Nat. Biotechnol. 31, 833–838 (2013).

    CAS  Article  Google Scholar 

  4. Lin, S., Ewen-Campen, B., Ni, X., Housden, B.E. & Perrimon, N. Genetics 201, 433–442 (2015).

    CAS  Article  Google Scholar 

  5. Maeder, M.L. et al. Nat. Methods 10, 977–979 (2013).

    CAS  Article  Google Scholar 

  6. Champer, J., Buchman, A. & Akbari, O.S. Nat. Rev. Genet. 17, 146–159 (2016).

    CAS  Article  Google Scholar 

  7. Nissim, L., Perli, S.D., Fridkin, A., Perez-Pinera, P. & Lu, T.K. Mol. Cell 54, 698–710 (2014).

    CAS  Article  Google Scholar 

  8. Xie, K., Minkenberg, B. & Yang, Y. Proc. Natl. Acad. Sci. USA 112, 3570–3575 (2015).

    CAS  Article  Google Scholar 

  9. Port, F., Muschalik, N. & Bullock, S.L. G3 (Bethesda) 5, 1493–1502 (2015).

    CAS  Article  Google Scholar 

  10. Xue, Z. et al. G3 (Bethesda) 4, 2167–2173 (2014).

    Article  Google Scholar 

  11. Port, F., Chen, H.-M., Lee, T. & Bullock, S.L. Proc. Natl. Acad. Sci. USA 111, E2967–E2976 (2014).

    CAS  Article  Google Scholar 

  12. Zetsche, B. et al. Cell 163, 759–771 (2015).

    CAS  Article  Google Scholar 

  13. Hur, J.K. et al. Nat. Biotechnol. 34, 807–808 (2016).

    CAS  Article  Google Scholar 

  14. Kim, D. et al. Nat. Biotechnol. 34, 863–868 (2016).

    CAS  Article  Google Scholar 

  15. Kim, Y. et al. Nat. Biotechnol. 34, 808–810 (2016).

    CAS  Article  Google Scholar 

  16. Kleinstiver, B.P. et al. Nat. Biotechnol. 34, 869–874 (2016).

    CAS  Article  Google Scholar 

  17. Moreno-Mateos, M.A. et al. Nat. Methods 12, 982–988 (2015).

    CAS  Article  Google Scholar 

  18. Kleinstiver, B.P. et al. Nature 529, 490–495 (2016).

    CAS  Article  Google Scholar 

  19. Slaymaker, I.M. et al. Science 351, 84–88 (2016).

    CAS  Article  Google Scholar 

  20. Noble, C. et al. Preprint at bioRxiv (2016).

  21. Dang, Y. et al. Genome Biol. 16, 280 (2015).

    Article  Google Scholar 

  22. Wang, J.-W., Beck, E.S. & McCabe, B.D. PLoS One 7, e42102 (2012).

    CAS  Article  Google Scholar 

  23. Pfeiffer, B.D., Truman, J.W. & Rubin, G.M. Proc. Natl. Acad. Sci. USA 109, 6626–6631 (2012).

    CAS  Article  Google Scholar 

  24. Mali, P. et al. Science 339, 823–826 (2013).

    CAS  Article  Google Scholar 

  25. Port, F. et al. Nat. Cell Biol. 10, 178–185 (2008).

    CAS  Article  Google Scholar 

  26. Schindelin, J. et al. Nat. Methods 9, 676–682 (2012).

    CAS  Article  Google Scholar 

Download references


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.)).

Author information

Authors and Affiliations



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.

Ethics declarations

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.

Supplementary information

Supplementary Text and Figures

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

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing