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Nat Methods. 2015 Jul;12(7):664-70. doi: 10.1038/nmeth.3433. Epub 2015 Jun 1.

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

Author information

1
1] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA. [3] Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, USA.
2
1] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA. [3] Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, USA. [4] Department of Pathology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA. [5].

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.

PMID:
26030444
PMCID:
PMC4821475
DOI:
10.1038/nmeth.3433
[Indexed for MEDLINE]
Free PMC Article

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