PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation

Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these prespacer substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 spe-cifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation com-plex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.

Automated summary

Adaptation in CRISPR-Cas systems provides immunity against mobile genetic elements for bacteria and archaea. This study reveals the cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex, which plays a crucial role in the selection and processing of DNA segments containing PAM sequences. The structures demonstrate how Cas4 reads bases within the PAM sequence and how its interactions with Cas1 and Cas2 activate its endonuclease activity.