Article ; Online: Programmable RNA recognition and cleavage by CRISPR/Cas9.
2014 Volume 516, Issue 7530, Page(s) 263–266
Abstract: ... context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition ... The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA ... to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate ...
| 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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| MeSH term(s) | Base Sequence ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/physiology ; Cell Extracts ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/chemistry ; DNA/genetics ; DNA/metabolism ; Genetic Engineering/methods ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics ; HeLa Cells ; Humans ; Nucleotide Motifs ; Oligonucleotides/chemistry ; Oligonucleotides/genetics ; Oligonucleotides/metabolism ; RNA/chemistry ; RNA/genetics ; RNA/metabolism ; RNA, Guide, CRISPR-Cas Systems/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; RNA, Messenger/genetics ; RNA, Messenger/isolation & purification ; RNA, Messenger/metabolism ; Substrate Specificity |
| Chemical Substances | CRISPR-Associated Proteins ; Cell Extracts ; Oligonucleotides ; RNA, Guide, CRISPR-Cas Systems ; RNA, Messenger ; RNA (63231-63-0) ; DNA (9007-49-2) ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) (EC 1.2.1.12) |
| Language | English |
| Publishing date | 2014-09-28 |
| Publishing country | England |
| Document type | Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. |
| ZDB-ID | 120714-3 |
| ISSN | 1476-4687 ; 0028-0836 |
| ISSN (online) | 1476-4687 |
| ISSN | 0028-0836 |
| DOI | 10.1038/nature13769 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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