Amplification-Free Dual-Blocking Autocatalytic CRISPR-Cascade for Atto-Molar DNA Detection with Low Nonspecific Signal

Amplification-free CRISPR diagnostics are often presented as simple positive feedback circuits, but most existing systems treat leakage, defined as target independent background activation that arises when blocked CRISPR components spontaneously form active ribonucleoprotein (RNP), as an unavoidable side effect rather than as a designable property. In particular, prior work has not explicitly accounted for two key sources of background signal in autocatalytic assays: Cas driven unwinding of blocked constructs and transient breathing of nucleic acid duplexes that intermittently expose trigger sites. Our study directly analyzes these leakage pathways in the context of switchable-cage-gRNA (scgRNA) and Cascade probe design and shows that blocking a single component is fundamentally vulnerable to both enzyme-driven strand invasion and equilibrium breathing. By contrast, we introduce a dual-blocking strategy in which both the guide RNA and the trigger DNA are gated. We further add a decoy guide RNA that competes for Cas12a. This multi-layer architecture demonstrates that robust amplification-free operation requires several coordinated barriers rather than a single switch, providing a new design principle for constructing self-amplifying CRISPR circuits with low background and robust signal-to-noise ratio.