Cas9 specifies functional viral targets during CRISPR–Cas adaptation

Robert Heler, Poulami Samai, Joshua W. Modell, Catherine Weiner, Gregory W. Goldberg, David Bikard () and Luciano A. Marraffini ()
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Robert Heler: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA
Poulami Samai: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA
Joshua W. Modell: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA
Catherine Weiner: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA
Gregory W. Goldberg: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA
David Bikard: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA
Luciano A. Marraffini: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA

Nature, 2015, vol. 519, issue 7542, 199-202

Abstract: 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.

Date: 2015
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DOI: 10.1038/nature14245

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