A Quantitative Crispr-Based Molecular Diagnostic System using Trans-Cleavage Kinetic Modeling for Accurate Viral Load Estimation

Abstract

Introduction: CRISPR-based molecular diagnostics have transformed nucleic acid detection but remain predominantly qualitative, limiting their utility for clinical viral load monitoring. Precise quantification is usually restricted to qPCR or digital assays that require complex hardware. Aim: To develop a quantitative CRISPR diagnostic framework that infers viral load from Cas trans-cleavage kinetics in bulk reactions using mechanistic modeling and plate-level calibration. Methodology: We modeled Cas12/Cas13 trans-cleavage with a Michaelis–Menten framework linking fluorescence time courses to an effective active enzyme concentration proportional to input viral copies after isothermal amplification. A calibration pipeline uses internal standards to fit kinetic parameters, map initial reaction velocity to viral load, and propagate uncertainty. Simulation studies were performed across realistic ranges of k_cat, K_M, reporter concentration, and instrument noise. Results: Simulations indicate that early-time fluorescence slopes robustly encode viral load over 10²–10⁶ copies per reaction with mean absolute error below ~0.25 log₁₀, approaching qPCR and digital CRISPR performance without partitioning. The framework remained stable under moderate assay variability when plate-specific calibration was applied. Conclusion: Trans-cleavage kinetic modeling can convert standard CRISPR assays into quantitative viral load tests using simple fluorescence hardware, providing a practical route toward portable, clinically meaningful CRISPR diagnostics.

Keywords: CRISPR diagnostics, Cas12, Cas13, Trans-cleavage kinetics, Michaelis–Menten, Viral load quantification.