The function of the ring-shaped protein Csn2 is reminiscent of the sliding clamp in DNA replication. Repurposing of #Cas9 nuclease by the #CRISPR adaptation machinery for prespacer selection characterized here demonstrates #Cas9 plasticity.
We propose a hypothetical spacer acquisition mechanism in type II-A CRISPR-Cas systems, consisting of prespacer selection by the Cas9-Cas1-Cas2-Csn2 supercomplex, and its step-wise hand-off to a Cas1-Cas2 #integrase.
We have determined the #cryoEM structure of the Csn2 - #integrase subcomplex forming the Interface 4. Interface 4 mutations abolished acquisition of new spacers in the native S. thermophilus host.
The catalytically inactive supercomplex may employ the alternative Cas1-Csn2 interface (Interface 4) to recruit the catalytic Cas1-Cas2 #integrase, or capture the mobile part of the Cas1-Cas2 #integrase that makes part of the supercomplex.
The supercomplex comprises 3 protein-protein interfaces: 1 (Cas1-Csn2), 2 (Cas9-Cas1), and 3 (Cas9-Csn2). Interface mutations abolished acquisition of new spacers in the native S. thermophilus host.
Upon PAM recognition the DNA-scanning supercomplex adopts the 'docked' conformation. Its further rearrangement via displacement of #Cas9 REC2 domain yields the 'locked' supercomplex with straight DNA.
Here, we present #cryoEM structures of the type II-A prespacer selection supercomplex consisting of #Cas9 RNP, Cas1-Cas2 #integrase fragment and Csn2 ring in the DNA-scanning and two different PAM-bound configurations.
In type II-A #CRISPR-Cas systems acquisition of new spacers involves the effector nuclease #Cas9, which forms a ‘supercomplex’ with the Cas1-Cas2 #integrase and the ring-shaped Csn2 protein. The supercomplex structure and the role of Csn2 remain unknown.
'Structural insights into Cas9-mediated prespacer selection in CRISPR-Cas adaptation' Check out our preprint on #bioRxiv on the #Cas9-Cas1-Cas2-Csn2 supercomplex involved in #CRISPR spacer acquisition: www.biorxiv.org/cgi/content/... Thread below.
