Thermodynamically programmed one-pot CRISPR platform for point-of-care SNP genotyping

One-pot CRISPR diagnostics face a fundamental incompatibility: nucleic acid amplification requires rapid target accumulation, whereas CRISPR activation irreversibly consumes those substrates, destabilizing reaction kinetics. Existing strategies rely on empirical parameter balancing or external staging but lack an intrinsic mechanism to enforce reaction order within a single reactor. Here we introduce thermodynamic encoding as a molecular design principle that programs reaction order directly into DNA primers, enabling autonomous, threshold-gated activation of CRISPR only after sufficient amplicon accumulated. By embedding a defined free-energy differential between competing primers, the system evolves through two kinetically ordered amplification regimes, decoupling amplification from CRISPR signal transduction without physical separation or external triggers. This architecture relocates PAM dependence from native genomic targets to primer-encoded design, enabling detection of otherwise inaccessible loci while preserving single-nucleotide discrimination. An ordinary differential equation model captures the threshold behavior and establishes a predictable framework for primer design. Building on this principle, we develop Thermodynamically Encoded Molecular Programming for One-pot diagnostics (TEMPO), which achieves attomolar sensitivity within 30 min and enables sequencing-concordant SNP genotyping and pathogen detection in a single-step microfluidic format.