BaCas12a3 represents a new subtype of type V CRISPR effector with collateral activity toward tRNA.

The CRISPR-Cas12 family encompasses diverse RNA-guided nucleases with both DNA- and RNA-targeting subtypes. They can trigger antiviral activities through either direct elimination of invading nucleic acids or activating broad collateral cleavage to induce abortive infection. Here, we report a novel type V CRISPR effector BaCas12a3 that causes growth inhibition through a unique tRNA-cleavage mechanism. Plasmid interference and western blot assays showed that BaCas12a3 induces host growth arrest without DNA damage response, suggestive of the absence of double-strand DNA breaks. Indeed, biochemical characterization of the BaCas12a3-crRNA ribonucleoprotein unraveled that the effector is an RNA-activating nuclease that cleaves the 3' terminal CCA of tRNAs. Cryo-EM structures of BaCas12a3 reveal a conserved bilobed architecture featuring a unique tRNA-loading domain (tRLD) adjacent to the RuvC catalytic center. Structural and mutagenesis analyses show that the tRLD domain, together with a zinc ribbon domain, form a gated substrate groove. Target RNA binding induces conformational changes that open the groove and expose the RuvC active site, enabling specific tRNA 3' end cleavage while preventing other non-specific degradation. Our findings identify the tRLD domain aside the RuvC active site responsible for the tRNA recognition in BaCas12a3, expanding the functional diversity of CRISPR immunity.