DNA assisting CRISPR-Cas13a activation regulating assay for detection of DNA mutation and miRNAs

Cas13a is an RNA-targeting effector system with exceptional sensitivity, essential for applications such as mutation discrimination, RNA biomarker quantification and dynamic monitoring. However, precise control over its activation kinetics and thermodynamics remains a fundamental challenge, limiting its use in nucleic acid detection. Here, we present the DNA-Assisting CRISPR-Cas13a Activation Regulating (DACAR) Assay, which employs a single-stranded DNA blocker to occupy the crRNA spacer. Through programmable toehold-mediated strand displacement (TMSD) and toehold-exchange (TE) reactions, DACAR is able to control the thermodynamics and kinetics of Cas13a activation at two different levels. Systematic modulation of toehold length and direction yields a tunable cleavage rate gradient, while TE-based free-energy tuning facilitates an ON/OFF gating mode. For single-nucleotide variant (SNV) discrimination, DACAR-TE amplifies thermodynamic mismatch effects, achieving a discrimination factor of 162.4 and a detection limit of 0.05% mutant abundance for the KRAS G12D mutation. The Switch-DACAR system also enables sequential detection of miR-155 and miR-21 in a single tube, with a detection limit of 10 pM. To sum up, DACAR provides a unified, programmable framework for precise molecular-level control of Cas13a, advancing its potential in diagnostics and molecular logic.