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bioRxiv · 10.64898/2026.06.23.733943

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

Zhao, C.·Xu, B.·Huang, X.·Han, X.·Xie, S.·Li, X.·Han, J.·Wu, D.·Li, S.·Zhao, S.
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Abstract

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.

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Zhao, C., Xu, B., Huang, X., Han, X., Xie, S., Li, X., Han, J., Wu, D., Li, S., Zhao, S.. 2026-06-24. Catalytic rewiring of RuvC-II catalytic site activates trans-cleavage in Fanzor2. https://doi.org/10.64898/2026.06.23.733943

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