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Nature. 2014 Dec 11;516(7530):263-6. doi: 10.1038/nature13769. Epub 2014 Sep 28.

Programmable RNA recognition and cleavage by CRISPR/Cas9.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
2
Department of Chemistry, University of California, Berkeley, California 94720, USA.
3
1] Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA [2] Department of Agricultural and Biological Engineering, University of Florida, Gainesville, Florida 32611, USA.
4
1] Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA [2] Department of Chemistry, University of California, Berkeley, California 94720, USA [3] Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA [4] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.

Abstract

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

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PMID:
25274302
PMCID:
PMC4268322
DOI:
10.1038/nature13769
[Indexed for MEDLINE]
Free PMC Article

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