RNA-triggered CRISPR-Cas12a2 Preferentially and Cooperatively Cleaves Collateral DNA

CRISPR-Cas systems often rely on collateral cleavage activities against nucleic-acid substrates to combat recognized mobile genetic elements. Of the associated RNA-guided Cas effector nucleases, Cas12a2 stands out as the only known example exhibiting RNA-triggered collateral cleavage of three distinct substrates: single-stranded (ss)RNA, ssDNA, and double-stranded (ds)DNA. However, little is known about the underlying mechanisms of collateral cleavage, including which of these activities dominates during the Cas12a2-mediated immune response. Here, we show that Cas12a2 cleaves collateral DNA substrates over RNA substrates, even when RNA substrates are more abundant. This preference relies on a positive cooperativity mechanism that requires the four "aromatic clamp" residues that stabilize unwound and distorted dsDNA in the RuvC nuclease active site. Leveraging the cleavage preference for collateral DNA, we demonstrate that RNA-activated Cas12a2 can cleave a ssDNA probe in the presence of high concentrations of non-target RNA, while the RNA-targeting Cas13a cannot. This work thus reveals how Cas12a2 decides amongst different collateral cleavage substrates, with immediate implications for understanding Cas12a2-based immunity, improving molecular diagnostics, and laying a mechnastic foundation for future Cas12a2-based technologies.