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

Next-generation sequencing offers highly multiplexed and accurate detection of nucleic acid sequences but at the expense of complex workflows and high input requirements. The ease of use of CRISPR-Cas12 assays is attractive and may enable highly accurate detection of sequences implicated in, for example, cancer pathogenic variants. CRISPR assays often employ end-point measurements of Cas12 trans-cleavage activity after Cas12 activation by the target; however, end point-based methods can be limited in accuracy and robustness by arbitrary experimental choices. To overcome such limitations, we develop and demonstrate here an accurate assay targeting a mutation of the epidermal growth factor gene implicated in lung cancer (exon 19 deletion). The assay is based on characterizing the kinetics of Cas12 trans-cleavage to discriminate the mutant from wild-type targets. We performed extensive experiments (780 reactions) to calibrate key assay design parameters, including the guide RNA sequence, reporter sequence, reporter concentration, enzyme concentration, and DNA target type. Interestingly, we observed a competitive reaction between the target and reporter molecules that has important consequences for the design of CRISPR assays, which use preamplification to improve sensitivity. Finally, we demonstrate the assay on 18 tumor-extracted amplicons and 100 training iterations with 99% accuracy and discuss discrimination parameters and models to improve wild type versus mutant classification.

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.