Cas9 specifies functional viral targets during CRISPR-Cas adaptation

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 guides that identify the viral targets (protospacers) of the Cas9 nuclease. Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of an NGG protospacer adjacent motif (PAM) sequence immediately downstream of the target. It is not known if and how viral sequences with the correct PAM are chosen as new spacers. Here we show that Cas9 specifies functional PAM sequences 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 the prokaryotic immunological memory.