Structural and Mechanistic Basis of PAM-Dependent Spacer Acquisition in CRISPR-Cas Systems

Bacteria acquire memory of viral invaders by incorporating invasive DNA sequence elements into the host CRISPR locus, generating a new spacer within the CRISPR array. We report on the structures of Cas1-Cas2-dual-forked DNA complexes in an effort toward understanding how the protospacer is sampled prior to insertion into the CRISPR locus. Our study reveals a protospacer DNA comprising a 23-bp duplex bracketed by tyrosine residues, together with anchored flanking 3′ overhang segments. The PAM-complementary sequence in the 3′ overhang is recognized by the Cas1a catalytic subunits in a base-specific manner, and subsequent cleavage at positions 5 nt from the duplex boundary generates a 33-nt DNA intermediate that is incorporated into the CRISPR array via a cut-and-paste mechanism. Upon protospacer binding, Cas1-Cas2 undergoes a significant conformational change, generating a flat surface conducive to proper protospacer recognition. Here, our study provides important structure-based mechanistic insights into PAM-dependent spacer acquisition. [Display omitted] •The dual-forked protospacer is integrated via a cut-and-paste mechanism•Architecture of Cas1-Cas2 predetermines length of newly acquired spacer•Cas1a recognizes PAM-complementary sequence via sequence-specific interactions•Cas1-Cas2 undergoes a conformational change upon protospacer DNA binding Cas1 and Cas2 select an invading DNA sequence, termed protospacer, for insertion into the CRISPR locus of the host cell. The structure of the Cas1-Cas2-protospacer DNA complex reveals the dual-forked nature of the protospacer, explains how the protospacer is selected, and identifies how protospacer length is predetermined.