title: Programmable RNA recognition and cleavage by CRISPR/Cas9.
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.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Mitchell R
authors.initials: MR
authors.lastname: O'Connell
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Oakes
authors.affiliation: Department of Chemistry, University of California, Berkeley, California 94720, USA.
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Sternberg
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Alexandra
authors.initials: A
authors.lastname: East-Seletsky
authors.affiliation: 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.
authors.forename: Matias
authors.initials: M
authors.lastname: Kaplan
authors.affiliation: 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.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
mesh_terms: D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000502:physiology*; D002457:Cell Extracts; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D059372:Nucleotide Motifs; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D064130:CRISPR-Associated Proteins; D002457:Cell Extracts; D009841:Oligonucleotides; D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)