Detection and Discrimination of Single Nucleotide Polymorphisms by Quantification of CRISPR-Cas Catalytic Efficiency

Abstract

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.

Design and Evaluation of a Robust CRISPR Kinetic Assay for Hot-Spot Genotyping

“…Some past studies have incorrectly reported that such a PAM is required for Cas12a activation for dsDNA targets, 38,39 but subsequent work has shown that PAM makes only a limited contribution to DNA unwinding. 18,40 Experimental Evaluation of gRNA and Reporter Selection. Example kinetic measurements are listed in Figure 1c.…”

Rapid and Sensitive Detection of Fungicide-Resistant Crop Fungal Pathogens Using an Isothermal Amplification Refractory Mutation System

“…A DNA strand modified with 6-carboxyfluorescein (6-FAM) at the 5′ end and black hole quencher 1 (BHQ1) at the 3′ end serves as the reporter of Cas12a. When the reporter is cut off via Cas12a activated by the RPA amplicon, the distance between 6-FAM and BHQ1 increases and the fluorescence from 6-FAM could be recovered. , The assay based on primer extension and CRISPR/Cas ensures reliable discrimination of single-nucleotide mutations in the fungal genome. Due to the high amplification efficiency of RPA, iARMS yields a high sensitivity for detecting fungi, and the detection processes could proceed at a constant temperature.…”

CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern

“…Although targeting mutations located in the PAM-proximal region has improved the specificity of CRISPR-Cas12a for the differentiation of SARS-CoV-2 and its variants, the ability of CRISPR-Cas12a to differentiate single nucleotide polymorphisms (SNPs) still needs to be further improved. Single-nucleotide mutation does not always lead to a decrease in the signal at a particular time point [ 141 ]. In a test covering all 60 possible mutations within a 20 nt protospacer, the mutant can even have a higher signal than the wild-type at one time point [ 141 ].…”

“…Single-nucleotide mutation does not always lead to a decrease in the signal at a particular time point [ 141 ]. In a test covering all 60 possible mutations within a 20 nt protospacer, the mutant can even have a higher signal than the wild-type at one time point [ 141 ]. An alternative strategy was to use the kinetics parameters for variant discrimination, because all these variants exhibited a lower catalytic efficiency ( k cat /K M ) than that of the wild-type [ 141 ].…”