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

Autocatalytic CRISPR architecture offers amplification-free nucleic acid detection by directly linking target recognition to self-reinforcing ribonucleoprotein (RNP) generation. However, spontaneous background activation remains a key barrier, because strand invasion or unwinding events can initiate unintended amplification and diminish assay specificity. Here, we introduce a dual-blocking CRISPR-Cascade design that independently cages both the guide RNA and trigger DNA, establishing an intrinsic AND gate to raise the effective kinetic barrier for unintended RNP formation. This strategy suppresses leakage by approximately 3- to 18-fold relative to single blocking configurations in full Cascade reactions, while preserving rapid detection (10 min), achieving single-copy sensitivity, and enabling quantitative detection. When paired with a competitive guide RNA decoy, the system further reduces background signals without affecting true target detection. Finally, we demonstrate robust Methicillin-resistant Staphylococcus aureus (MRSA) detection from whole blood in under 40 minutes including the sample purification and extraction. These results establish dual-blocking as a generalizable molecular gating framework for constructing leakage-resistant, amplification-free CRISPR systems suitable for rapid and decentralized diagnostics. Significance: 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.