Credit: NPG

The CRISPR–Cas (clustered, regularly interspaced short palindromic repeats–CRISPR-associated proteins) adaptive immune systems of archaea and bacteria function in the elimination of foreign nucleic acids derived from invading pathogens and plasmids. In most systems, mature CRISPR RNAs (crRNAs) guide the Cas protein-mediated cleavage of invading target sequences; however, in the type II CRISPR–Cas system, dual-guide RNAs (a crRNA and a trans-acting RNA (tracrRNA)) are needed to target the endonuclease Cas9 to specific DNA sites, where it mediates double-strand breaks. Two new structural studies now report that a conformational switch in Cas9 leads to its catalytic activation and highlight the role of the protospacer adjacent motif (PAM) in Cas9 target recognition and cleavage.

Jinek et al. solved the crystal structures of Streptococcus pyogenes and Actinomyces naeslundii Cas9 and showed that these enzymes contain a conserved catalytic core that comprises a nuclease lobe (for DNA cleavage) and a variable, α-helical lobe. Two clefts located in the α-helical and nuclear lobes were also identified, which bind to guide RNAs and target sequences, respectively. Single-particle electron microscopy reconstructions showed that, in the apo state, Cas9 adopts a conformation that is unable to bind and cleave target DNA; however, association of the crRNA–tracrRNA duplex with Cas9 induced the reorientation of the two structural lobes to form a central channel that can accommodate the target sequence. Thus, loading of the crRNA–tracrRNA duplex is required for the activation of Cas9.

It has previously been suggested that a PAM located in the target sequence is crucial for Cas9 activity. In the second study, Sternberg et al. used single-molecule imaging to show that the Cas9–guide RNA complex forms a long-lived binding interaction with target sites that contain a PAM, whereas transient binding was observed at non-target sequences and at complementary sequences that lacked PAMs. These results suggest that Cas9 quickly dissociates from non-target sequences but binds more strongly in the presence of a PAM to search the flanking DNA regions for potential guide-RNA complementarity. Using a cleavage-competitor assay, the authors also showed that at complementary targets, guide RNA–target DNA heteroduplex formation is initiated at the PAM and that R-loop formation proceeds via the sequential and directed unwinding of the double-stranded DNA. Finally, DNA cleavage was dependent on the PAM, which suggests that the PAM triggers Cas9 nuclease activity. The authors propose that this PAM dependency eliminates the potential for self-targeting, as matching DNA in the CRISPR array does not contain these motifs.

Together, these studies show that Cas9 contains a conserved catalytic core within a variable overall protein architecture and that Cas9 requires activation by the guide RNA; they also reveal the mechanistic basis for the ability of Cas9 to identify target sequences.