title: Cas9 specifies functional viral targets during CRISPR-Cas adaptation.
abstract: Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.
authors.affiliation: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Heler
authors.affiliation: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.
authors.forename: Poulami
authors.initials: P
authors.lastname: Samai
authors.affiliation: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.
authors.forename: Joshua W
authors.initials: JW
authors.lastname: Modell
authors.affiliation: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.
authors.forename: Catherine
authors.initials: C
authors.lastname: Weiner
authors.affiliation: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.
authors.forename: Gregory W
authors.initials: GW
authors.lastname: Goldberg
authors.affiliation: 1] Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA [2] Synthetic Biology Group, Institut Pasteur, 28 Rue du Dr. Roux, 75015 Paris, France.
authors.forename: David
authors.initials: D
authors.lastname: Bikard
authors.affiliation: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.
authors.forename: Luciano A
authors.initials: LA
authors.lastname: Marraffini
mesh_terms: D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000276:immunology; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics* / Q000276:immunology; D004279:DNA, Viral / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D059372:Nucleotide Motifs; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013211:Staphylococcus aureus; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology / Q000821:virology; D013379:Substrate Specificity
chemical_list: D064130:CRISPR-Associated Proteins; D004279:DNA, Viral