Alexandre Avaro

This Feature Article reviews engineering guidelines for the design of CRISPR assays, including experimentally validated theoretical models and recommendations for experimental research practice and reporting. First, the state of the art of CRISPR kinetics studies is reviewed. Then presented is a summary of the existence and persistence of widespread gross errors in reports of kinetic rate constants of CRISPR-Cas enzymes, as well as the fact that many CRISPR studies provide insufficient data to check for consistency or assess calibration. Proper experimental procedures including signal calibration are critical to the assessment, design, and future development of CRISPR kinetics assays and CRISPR diagnostics. This review then presents guidelines for the calibration of fluorescence-based CRISPR assays. Fluorescence is the most common detection modality, and incorrect calibration is implicated in high-profile, gross errors in the field. Also presented is a review of enzymatic kinetic rates and reporter molecule degradation as the major factor limiting CRISPR assay sensitivity. Lastly, progress in, and criticism of, microfluidic applications of CRISPR assays is summarized.

The development of microfluidic technologies has enabled chemical and biological analysis systems with increased functionality, complexity, and parallelization. These functionalities often drive the creation and control of complex and dynamic fluidic architectures. Introduced here is a class of microfluidic network based on isotachophoresis (ITP), an electrokinetic process that can extract and purify samples, selectively transport, mix, and aliquot (split) samples in a system with no moving parts. Presented is a theoretical framework to describe these networks. The framework relies on the coupling between a one-dimensional description of ITP and two-dimensional, transient graphs to describe the dynamic evolution of ITP networks. We leverage this framework to create numerical simulations of branched ITP circuits. We build, control, and experimentally study a variety of ITP networks. These systems automatically split and merge ITP zones, enabling complex sample manipulation with minimal external control. The model captures the experimentally observed sample dynamics. We demonstrate an example system where an ITP network is used to control and quantify parallel CRISPR-Cas enzymatic reactions. The methods described here are generally applicable to highly complex topologies and may offer a basis for easily reconfigurable, electric field-driven microfluidic systems. Networks generally offer broad potential for automated chemical and biochemical analysis and lab-on-a-chip integration.

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.