Large-scale structural rearrangements unleash indiscriminate nuclease activity of CRISPR-Cas12a2

Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided degradation of non-specific single-stranded (ss)RNA, ssDNA and double-stranded (ds)DNA upon recognition of a complementary RNA target, culminating in abortive infection (Dmytrenko 2022). Here, we report structures of Cas12a2 in binary, ternary, and quaternary complexes to reveal a complete activation pathway. Our structures reveal that Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft. Double-stranded DNA substrates are captured through duplex distortion and local melting, stabilized by pairs of ‘aromatic clamp’ residues that are crucial for dsDNA degradation and in <jats:italic>vivo</jats:italic> immune system function. Our work provides a structural basis for this unprecedented mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates.