Rarely acquired type II-A CRISPR-Cas spacers mediate anti-viral immunity through the targeting of a non-canonical PAM sequence.

The Streptococcus pyogenes type II-A CRISPR-Cas systems provides adaptive immunity through the acquisition of short DNA sequences from invading viral genomes, called spacers. Spacers are transcribed into short RNA guides that match regions of the viral genome followed by a conserved NGG DNA motif, known as the PAM. These RNA guides, in turn, are used by the Cas9 nuclease to find and destroy complementary DNA targets within the viral genome. While most of the spacers present in bacterial populations that survive phage infection target protospacers flanked by NGG sequences, there is a small fraction that target non-canonical PAMs. Whether these spacers originate through accidental acquisition of phage sequences and/or provide efficient defense is unknown. Here we found that many of them match phage target regions flanked by an NAGG PAM. Despite being scarcely present in bacterial populations, NAGG spacers provide substantial immunity in vivo and generate RNA guides that support robust DNA cleavage by Cas9 in vitro; with both activities comparable to spacers that target sequences followed by the canonical AGG PAM. In contrast, acquisition experiments showed that NAGG spacers are acquired at very low frequencies. We therefore conclude that discrimination against these sequences occurs during immunization of the host. Our results reveal unexpected differences in PAM recognition during the spacer acquisition and targeting stages of the type II-A CRISPR-Cas immune response.