CRISPR Enzyme Kinetics for Molecular Diagnostics

Abstract

CRISPR-diagnostic assays have gained significant interest in the last few years. This interest has grown rapidly during the current COVID-19 pandemic, where CRISPR-diagnostics have been frontline contenders for rapid testing solutions. This surge in CRISPR-diagnostic research prompts the following question: what exactly are the achievable limits of detection and associated assay times enabled by the kinetics of enzymes such as Cas12 and Cas13? To explore this question, we here present a model based on Michaelis–Menten enzyme kinetics theory applied to CRISPR enzymes. We use the model to develop analytical solutions for reaction kinetics and develop back-of-the-envelope criteria to validate and check for consistency in reported enzyme kinetic parameters. We applied our analyses to all studies known to us, which report Michaelis–Menten-type kinetic data for CRISPR-associated enzymes. These studies include all subtypes of Cas12 and Cas13 and orthologs. We found all but one study clearly violate at least two of our three rules and therefore present data that violate basic physical limits. We performed an experimental study of reaction kinetics of LbCas12a with both ssDNA and dsDNA activators and use these data to validate our model and its predicted scaling. The validated model is used to explore CRISPR reaction time scales and the degree of reaction completion for practically relevant target concentrations applicable to CRISPR-diagnostic assays. The results have broad implications for achievable limits of detection and assay times of emerging, amplification-free CRISPR-detection methods.

Definition of CRISPR Cas12a Trans-Cleavage Units to Facilitate CRISPR Diagnostics

“…Then, with the employment of a limited concentration of Cas12a and crRNA and varied concentrations of ssDNA reporters, trans -cleavage enzymatic kinetics for Cas12a were determined, including the values of v max , K m , and K cat ( Figure 4 and Supplementary Table 2 ). Noticeably, the constants of K m , K cat as well as the K cat / K m were miscalculated in a previous study ( Chen et al, 2018 ), which were much higher than the data in this study and another work ( Ramachandran and Santiago, 2021 ) and have been recently corrected ( Supplementary Table 3 ).…”

“…To precisely calculate the Cas12a trans -cleavage activity, the ssDNA reporter used was dual labeled with fluorophore and quencher (i.e., FQ-reporter), and the reaction was monitored by a fluorescence reader. To simplify the calculation, the trans -cleavage activity was calculated on the basis of the initial fluorescence growth rate v g ( v g = ΔF/Δt), following the recently published Michaelis-Menten model ( Ramachandran and Santiago, 2021 ).…”

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform

“…Despite the single-base resolution of Cas12, the kinetics of CRISPR enzyme for molecular diagnosis recently revealed that realistic LODs for Cas13/Cas12-based detection system were only 10 pM to 100 fM, regardless of the number of targets ( Ramachandran and Santiago, 2021 ). Thus, signal amplification is essential either upstream or downstream of CRISPR reactions to achieve high sensitivity ( Chen et al, 2020 ; Li et al, 2021a ; Qiao et al, 2021 ).…”

Applications of the versatile CRISPR‐Cas13 RNA targeting system

“…Again, adapting mobile phone cameras as detectors in simple detection set-ups increased the convenience of this approach on the field (Figure 3). However, the kinetic parameters and calculated limit of detection reported in this study have recently been challenged (Ramachandran & Santiago, 2021). Further implementations involve the detection of the herpes viruses BKV and CMV following F I G U R E 3 Nucleic-acid detection by Cas13 collateral cleavage.…”

“…It is thus possible that the collateral effect might escape detection due to the timing of samples collection, or validation methods used, and might be more prominent and easier to detect with plasmid-borne reporter systems, or when it occurs during complex developmental or differentiation processes. These uncertainties call for more detailed biochemical investigations of Cas13 enzymes kinetics, as addressed recently for the in vitro activities (Ramachandran & Santiago, 2021), and investigations of Cas13:crRNA:target RNA stabilities in different model systems. Essentially, the window of time in which the collateral effect can occur is largely unknown.…”