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Amyloid fibril formation is a hallmark of several protein misfolding diseases, including systemic hereditary amyloidosis (SHA), in which lysozyme aggregates into plaques, causing inflammation to various tissues. SHA is a rare disease with no current drug treatment options. In our efforts to identify potential therapeutics for SHA, we investigated the inhibitory effects of benzamidine (BEN) on the human lysozyme fibrillation (HLF). Multiple biophysical assays demonstrated BEN's ability to prevent the lysozyme fibrillation. Intrinsic fluorescence measurements highlighted BEN's interaction with human native lysozyme (HNL). We inferred that the binding mode of BEN to HNL through ITC, molecular docking, and molecular dynamics simulations, confirmed BEN's binding at the active site, near stretch-2 (residues 52-64). The circular dichroism (CD) analysis revealed alterations in the secondary structure of HNL and HLF upon BEN's treatment. In HLF, an increase in BEN's concentration resulted in a concentration-dependent reduction in β-sheet content. SEM and TEM analyses revealed reduced fibril formation and significant alterations in fibril morphology in the presence of BEN. Importantly, BEN exhibited no cytotoxic effects in HEK-293 cells. These results provide strong evidence of BEN's anti- amyloidogenic activity and offer a foundation for future drug development targeting lysozyme amyloidosis., Competing Interests: Declaration of competing interest “The authors declare that they have no competing interests.”, (Copyright © 2025. Published by Elsevier B.V.)

CRISPR-Cas12a is widely used for genome editing and biomarker detection since it can create targeted double-stranded DNA breaks and promote non-specific DNA cleavage after identifying specific DNA. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Francisella novicida Cas12a variant (FnoCas12a KD2P ) by introducing double proline substitutions (K969P/D970P) in a conserved helix called the bridge helix (BH). In this work, we used cryogenic electron microscopy (cryoEM) to understand the molecular mechanisms of BH-mediated activation of Cas12a. We captured five structures of FnoCas12a KD2P at different states of conformational activation. Comparison with wild-type (FnoCas12a WT ) structures unravels a mechanism where BH acts as a trigger that allosterically activates REC lobe movements by tracking the number of base pairs in the growing RNA-DNA hybrid to undergo a loop-to-helical transition and bending to latch onto the hybrid. The transition of the BH is coupled to the previously reported loop-to-helix transition of the "lid", essential for opening RuvC endonuclease, through direct interactions of residues of the BH and the lid. We also observe structural details of cooperativity of BH and "helix-1" of RuvC for activation, a previously proposed interaction. Overall, our study enables development of high-fidelity Cas12a and Cas9 variants by BH-modifications.