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 𝑘 𝑐𝑎𝑡 , 𝐾 𝑀 , and catalytic efficiency 𝑘 𝑐𝑎𝑡 /𝐾 𝑀 . 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.

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 (K M ) and apparent catalytic efficiency (k cat * /K M ). 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 k cat * /K M measured for WT is 130-fold greater than the lowest k cat * /K M among all 60 measured SNPs (compared to a 4.8-fold for endpoint fluorescence of the same SNP). K M 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.

The specificity of CRISPR-Cas12 assays is attractive for the detection of single nucleotide polymorphisms (SNPs) implicated in, e.g., 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 shifts in the Cas12 trans-cleavage substrate affinity (KM) and apparent catalytic efficiency . To address endpoint-based assay limitations, we then develop an assay based on the quantification of Michalis-Menten parameters and apply this assay to a 20-base pair WT target of the SARS-CoV-2 E gene. We find that the measured for WT is 130-fold greater than the lowest among all 60 measured SNPs (compared to a 4.8-fold for endpoint fluorescence of the same SNP). KM also offers strong ability to distinguish SNPs, varies 27-fold over all the cases, and is insensitive to target concentration. Lastly, we point out trends among kinetic rates and SNP base and location within the CRISPR-Cas12 targeted region.