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Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display

Nature Methods volume 12, pages 664670 (2015) | Download Citation

Abstract

Noncoding RNAs play diverse roles throughout biology and exhibit broad functional capacity. To investigate and harness these capabilities, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-Display (CRISP-Disp), a targeted localization method that uses Cas9 to deploy large RNA cargos to DNA loci. We demonstrate that functional RNA domains up to at least 4.8 kb long can be inserted in CRISPR guide RNA at multiple points, allowing the construction of Cas9 complexes with protein-binding cassettes, artificial aptamers, pools of random sequences and natural long noncoding RNAs. A unique feature of CRISP-Disp is the multiplexing of distinct functions at multiple targets, limited only by the availability of functional RNA motifs. We anticipate the use of CRISP-Disp for ectopically targeting functional RNAs and ribonucleoprotein (RNP) complexes to genomic loci.

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Acknowledgements

We thank L. Cong and F. Zhang for TALE plasmids and advice on reporter construct design; Y. Sancak, C. Fulco, S. Donovan and M. Morse for their general technical assistance; G. Kenty for his help with luminometry; D. Hendrickson and D. Tenen for their RIP expertise; C. Gerhardinger and C. Daly for their support with deep sequencing; and M. Tabebordbar, J. LaVecchio, S. Ionescu and M. Sauvageau for their assistance with FACS. We are grateful to all members of the Rinn laboratory for their thoughtful discussions and critiques. This work was supported by US National Institutes of Health grant P01GM099117.

Author information

Affiliations

  1. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.

    • David M Shechner
    • , Ezgi Hacisuleyman
    • , Scott T Younger
    •  & John L Rinn
  2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

    • David M Shechner
    • , Ezgi Hacisuleyman
    • , Scott T Younger
    •  & John L Rinn
  3. Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, USA.

    • David M Shechner
    • , Ezgi Hacisuleyman
    • , Scott T Younger
    •  & John L Rinn
  4. Department of Pathology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

    • John L Rinn
    • John L Rinn

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Contributions

D.M.S. designed and performed experiments; E.H. designed and performed microscopy experiments (Figs. 4c,d and Supplementary Figs. 13 and 14); S.T.Y. assisted with computational analysis (Fig. 3d and Supplementary Figs. 5 and 10); and J.L.R. directed research. D.M.S. and J.L.R. wrote the manuscript.

Competing interests

The authors declare competing financial interests. D.M.S. and J.L.R. have filed for patents concerning the design and expression of extended sgRNAs to expand the repertoire of dCas9 function.

Corresponding author

Correspondence to John L Rinn.

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    Supplementary Figures 1–14, Supplementary Tables 1–6 and Supplementary Notes 1–3

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DOI

https://doi.org/10.1038/nmeth.3433

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