Degradation of Reporter Molecules Imposes a Fundamental Limit of Detection on CRISPR Diagnostics

The sensitivity of CRISPR-Cas systems used for molecular diagnostics remains a major bottleneck in the adoption of this technology. The vast majority of CRISPR-based assays use dually labeled, single-stranded reporters and fluorescence signal readouts to infer enzymatic activity and the presence (or absence) of target nucleic acid. The limit of detection of such assays is set by the kinetics of the Cas enzymes and a slow yet measurable increase in the fluorescence signal. We demonstrate here that the background signal and limits of detection of most assays are very likely limited by the degradation of reporter molecules. This degradation is dynamic and is not associated with enzymatic activity. We present theory and experiments to design and calibrate CRISPR assays. We introduce a new kinetic framework to account for the degradation of reporter molecules and derive a fundamental limit of detection for CRISPR-based assays. Our data show that Michaelis-Menten kinetics alone are insufficient to describe reporter (substrate) cleavage rates. The framework and techniques presented here should help reduce the frequency and magnitude of errors currently routinely made in quantifying CRISPR kinetics and interpreting CRISPR diagnostic fluorescence signals.