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Antiviral defense by type III CRISPR-Cas systems relies on two distinct activities of their effectors: the RNA-activated DNA cleavage and synthesis of cyclic oligoadenylate. Both activities are featured as indiscriminate nucleic acid cleavage and subjected to the spatiotemporal regulation. To yield further insights into the involved mechanisms, we reconstituted LdCsm, a lactobacilli III-A system in  Escherichia coli . Upon activation by target RNA, this immune system mediates robust DNA degradation but lacks the synthesis of cyclic oligoadenylates. Mutagenesis of the Csm3 and Cas10 conserved residues revealed that Csm3 and multiple structural domains in Cas10 function in the allosteric regulation to yield an active enzyme. Target RNAs carrying various truncations in the 3ʹ anti-tag were designed and tested for their influence on DNA binding and DNA cleavage of LdCsm. Three distinct states of ternary LdCsm complexes were identified. In particular, binding of target RNAs carrying a single nucleotide in the 3ʹ anti-tag to LdCsm yielded an active LdCsm DNase regardless whether the nucleotide shows a mismatch, as in the cognate target RNA (CTR), or a match, as in the noncognate target RNA (NTR), to the 5′ tag of crRNA. In addition, further increasing the number of 3ʹ anti-tag in CTR facilitated the substrate binding and enhanced the substrate degradation whereas doing the same as in NTR gradually decreased the substrate binding and eventually shut off the DNA cleavage by the enzyme. Together, these results provide the mechanistic insights into the allosteric activation and repression of LdCsm enzymes.

Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase