Mechanism for Cas4-assisted directional spacer acquisition in CRISPR-Cas

Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array.sup.1. Spacer insertion is carried out by the Cas1-Cas2 integrase complex.sup.2-4. A substantial fraction of CRISPR-Cas systems use a Fe-S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM).sup.5,6 and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens, in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3'-overhang trigger Cas4/Cas1-Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This 'molecular constipation' prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1-Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality. Structures of the Cas4-Cas1-Cas2 complex from Geobacter sulfurreducens show that a 3'-overhang in the protospacer adjacent motif is required for complex assembly and spacer insertion into the CRISPR array.
Author(s): Chunyi Hu [sup.1] , Cristóbal Almendros [sup.2] [sup.3] , Ki Hyun Nam [sup.4] , Ana Rita Costa [sup.2] [sup.3] , Jochem N. A. Vink [sup.2] [sup.3] , Anna C. [...]