Catalytic rewiring of RuvC-II catalytic site activates trans -cleavage in Fanzor2

Fanzors are eukaryotic RNA-guided endonucleases that mediate programmable cis -cleavage in eukaryotic cells, but their potential target-induced collateral ( trans ) cleavage activity remains largely unexplored. Here, we show that catalytic rewiring of the RuvC-II catalytic site activates robust trans -cleavage activity in Acanthamoeba polyphaga mimivirus Fanzor2 (ApmFz2). The representative variant ApmFz2-EP displayed DNA– and RNA-triggered trans -cleavage, attenuated cis -cleavage, minimal TAM dependence, and activation by as few as seven nucleotides of guide-target complementarity. Mechanistic analyses indicate that relieving steric constraints surrounding the conserved alternative glutamate within the RuvC-II activates trans -cleavage and reshapes target-recognition specificity. Coupling ApmFz2-EP with nucleic acid amplification enabled FINDER, a Fanzor-based diagnostic platform for sensitive pathogen detection and broad detection across genetically diverse target subtypes. In addition, the mismatch-sensitive ApmFz2-EA variant enabled specific single-nucleotide variant (SNV) genotyping. Together, this work expands the functional scope of Fanzors and identifies catalytic-center engineering as a strategy for developing compact, programmable nucleic acid diagnostics.