Structural insights into Cas9-mediated prespacer selection in CRISPR-Cas adaptation. Gaizauskaite et al.

Spacer acquisition relies on the Cas1-Cas2 integrase and accessory proteins, which select DNA sequences flanked by the protospacer adjacent motif (PAM) and insert them into the CRISPR array. It has been shown that in type II-A systems selection of PAM-proximal prespacers is mediated by the effector nuclease Cas9, which forms a ‘supercomplex’ with the Cas1-Cas2 integrase and the Csn2 protein.

Here, we present cryo-electron microscopy structures of the type II-A prespacer selection supercomplex in the DNA-scanning and two distinct PAM-bound configurations, providing insights into the mechanism of Cas9-mediated prespacer selection in type II-A CRISPR-Cas systems. Repurposing of Cas9 by the CRISPR adaptation machinery for prespacer selection characterized here demonstrates Cas9 plasticity and expands our knowledge of the Cas9 biology.