Abstract
<jats:p>Tandem interspaced guide RNA (TIGR)-TIGR-associated (Tas) are RNA-guided defense systems, which use a dual-repeat or stem-loop tigRNA to direct a Tas dimer to DNA targets through RNA-DNA heteroduplex formation between both DNA strands. While previous work has shown the basic principles of DNA targeting, how the guide architecture influences target recognition and whether recognition and cleavage are coordinated across the two RNA-DNA heteroduplexes at a target site remain poorly understood. Here, we combine cryo-electron microscopy, biochemistry, and cell-based assays to investigate two TIGR-Tas effectors: the nuclease-lacking Peromyscus leucopus TasA (PlTasA), associated with a stem-loop tigRNA, and the nuclease-active Salicola phage TasH (SpTasH). Cryo-EM structures of PlTasA binary and ternary complexes reveal a dimeric scaffold similar to SpTasH and TaTasR with a distinct stem-loop tigRNA architecture and additional peripheral structural elements. Binding assays using DNA substrates containing local bubbles across the spacer-matching region show equivalent bubbles produced position- and spacer-dependent effects, indicating that target engagement is asymmetric in both PlTasA and SpTasH. Kinetic and cryo-EM analyses of SpTasH further reveal stepwise heteroduplex formation, with a partially engaged intermediate that undergoes substantial conformational rearrangements of the second protomer, preceding a fully paired state poised for catalytic activation. Cleavage of the two DNA strands occurs through a coordinated, slow process and productive cleavage requires stringent surveillance of both heteroduplexes. Together, these findings define a conserved, ordered mechanism of bipartite target recognition and activation shared by TIGR-Tas effectors, expanding our understanding of the molecular principles underlying programmable DNA targeting by TIGR-Tas systems.</jats:p>