A critical review of microfluidic systems for CRISPR assays.
Lab on a chip
Reviewed are nucleic acid detection assays that incorporate clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics and microfluidic devices and techniques. The review serves as a reference for researchers who wish to use CRISPR-Cas systems for diagnostics in microfluidic devices. The review is organized in sections reflecting a basic five-step workflow common to most CRISPR-based assays. These steps are analyte extraction, pre-amplification, target recognition, transduction, and detection. The systems described include custom microfluidic chips and custom (benchtop) chip control devices for automated assays steps. Also included are partition formats for digital assays and lateral flow biosensors as a readout modality. CRISPR-based, microfluidics-driven assays offer highly specific detection and are compatible with parallel, combinatorial implementation. They are highly reconfigurable, and assays are compatible with isothermal and even room temperature operation. A major drawback of these assays is the fact that reports of kinetic rates of these enzymes have been highly inconsistent (many demonstrably erroneous), and the low kinetic rate activity of these enzymes limits achievable sensitivity without pre-amplification. Further, the current state-of-the-art of CRISPR assays is such that nearly all systems rely on off-chip assays steps, particularly off-chip sample preparation.
View details for DOI 10.1039/d2lc00852a
View details for PubMedID 36601854
Uncertainty Quantification of Michaelis-Menten Kinetic Rates and Its Application to the Analysis of CRISPR-Based Diagnostics.
Angewandte Chemie (International ed. in English)
Michaelis-Menten kinetics is an essential model to rationalize enzyme reactions. The quantification of Michaelis-Menten parameters can be very challenging as it is sensitive to even small experimental errors. We here present a quantification of the uncertainty inherent to the experimental determination of kinetic rate parameters for enzymatic reactions. We study the influence of several sources of uncertainty and bias, including the inner filter effect, pipetting errors, number of points in the Michaelis-Menten curve, and flat-field correction. Using Monte Carlo simulations and analyses of experimental data, we compute typical uncertainties of [[EQUATION]] , [[EQUATION]] , and catalytic efficiency [[EQUATION]] . As a salient example, we analyze the extraction of such parameters for CRISPR-Cas systems. CRISPR diagnostics have recently attracted much interest and yet reports of these enzymatic kinetic rates have been highly unreliable and inconsistent.
View details for DOI 10.1002/anie.202209527
View details for PubMedID 36117459
Web-Based Open-Source Tool for Isotachophoresis.
View details for DOI 10.1021/acs.analchem.1c03925
View details for PubMedID 34788021
Detection and Discrimination of Single Nucleotide Polymorphisms by Quantification of CRISPR-Cas Catalytic Efficiency.
The specificity of CRISPR-Cas12 assays is attractive for the detection of single nucleotide polymorphisms (SNPs) implicated in, e.g., cancer and SARS-CoV-2 variants. Such assays often employ endpoint measurements of SNP or wild type (WT) activated Cas12 trans-cleavage activity; however, the fundamental kinetic effects of SNP versus WT activation remain unknown. We here show that endpoint-based assays are limited by arbitrary experimental choices (like used reporter concentration and assay duration) and work best for known target concentrations. More importantly, we show that SNP (versus WT) activation results in measurable kinetic shifts in the Cas12 trans-cleavage substrate affinity (KM) and apparent catalytic efficiency (kcat*/KM). To address endpoint-based assay limitations, we then develop an assay based on the quantification of Michaelis-Menten parameters and apply this assay to a 20 base pair WT target of the SARS-CoV-2 E gene. We find that the kcat*/KM measured for WT is 130-fold greater than the lowest kcat*/KM among all 60 measured SNPs (compared to a 4.8-fold for endpoint fluorescence of the same SNP). KM also offers a strong ability to distinguish SNPs, varies 27-fold over all the cases, and, importantly, is insensitive to the target concentration. Last, we point out trends among kinetic rates and SNP base and location within the CRISPR-Cas12 targeted region.
View details for DOI 10.1021/acs.analchem.2c03338
View details for PubMedID 36251847