Enzyme Kinetics and Detector Sensitivity Determine Limits of Detection of Amplification-Free CRISPR-Cas12 and CRISPR-Cas13 Diagnostics

Abstract

Interest in CRISPR-Cas12 and CRISPR-Cas13 detection continues to increase as these detection schemes enable the specific recognition of nucleic acids. The fundamental sensitivity limits of these schemes (and their applicability in amplification-free assays) are governed by kinetic rates. However, these kinetic rates remain poorly understood, and their reporting has been inconsistent. We quantify kinetic parameters for several enzymes (LbCas12a, AsCas12a, AapCas12b, LwaCas13a, and LbuCas13a) and their corresponding limits of detection (LoD). Collectively, we present quantification of enzyme kinetics for 14 guide RNAs (gRNAs) and nucleic acid targets for a total of 50 sets of kinetic rate parameters and 25 LoDs. We validate the self-consistency of our measurements by comparing trends and limiting behaviors with a Michaelis−Menten trans-cleavage reaction kinetics model. For our assay conditions, activated Cas12 and Cas13 enzymes exhibit trans-cleavage catalytic efficiencies between order 10 5 and 10 6 M −1 s −1 . For assays that use fluorescent reporter molecules (ssDNA and ssRNA) for target detection, the kinetic rates at the current assay conditions result in an amplification-free LoD in the picomolar range. The results suggest that successful detection of target requires cleavage (by an activated CRISPR enzyme) of the order of at least 0.1% of the fluorescent reporter molecules. This fraction of reporters cleaved is required to differentiate the signal from the background, and we hypothesize that this required fraction is largely independent of the detection method (e.g., endpoint vs reaction velocity) and detector sensitivity. Our results demonstrate the fundamental nature by which kinetic rates and background signal limit LoDs and thus highlight areas of improvement for the emerging field of CRISPR diagnostics.

Inconsistent treatments of the kinetics of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) impair assessment of its diagnostic potential

“…The latter assay used two LbuCas13a systems, a 400 nM reporter concentration, and a mobile phone camera for detection in 30 min. Given our own quantification of LbuCas13a kinetics in similar buffers (𝑘 /𝐾 of order 4E6 M -1 s -1 , Huyke et al 2022) we estimate that the latter work reports detection after cleaving less than about 0.0001% of the fluorophore-quencher reporters in the reaction.…”

“…The number of and variety among CRISPR assays without amplification is growing rapidly with microfluidic chip formats (Fozouni et al 2021;Bruch et al 2019) including droplet systems (Shinoda et al 2021;Ackerman et al 2020), lateral flow readout formats (Gootenberg et al 2018), and detection using multiple types of Cas enzymes per target (Liu et al 2021) Accepted manuscript however, makes estimates of the LOD of CRISPR-based enzyme kinetics parameters (Huyke et al 2022).…”

“…in absence of target). This important drawback of these enzymes very likely limits their LoD(Huyke et al 2022) and has been document by a variety of groups(Fozouni et al 2021;Shinoda et al 2021;Nalefski et al 2021;Nguyen, Smith, and Jain 2020;Yue et al 2021;Shan et al 2019;Palmier and Van Doren 2007;Huyke et al 2022).…”

Fast bioluminescent nucleic acid detection using one-pot isothermal amplification and dCas9-based split luciferase complementation

“…2C, D), represents a substantial improvement in sensitivity over prior bioluminescent nucleic acid sensors with LODs of ∼ 6 – 50 pM 17,18,22 . LUNAS is on par with fluorescent CRISPR Cas12- and Cas13-based methods excluding target pre-amplification, which feature LODs of ∼ 1 pM – 5 nM depending on the specific Cas12/Cas13 orthologs used, with recently reported sensitivities down to 166 fM only achieved by measuring for 1 – 2 hours 11,29,30 .…”

“…2C, D), represents a substantial improvement in sensitivity over prior bioluminescent nucleic acid sensors with LODs of ~ 6 -50 pM 17,18,22 . LUNAS is on par with fluorescent CRISPR Cas12-and Cas13-based methods excluding target pre-amplification, which feature LODs of ~ 1 pM -5 nM depending on the specific Cas12/Cas13 orthologs used, with recently reported sensitivities down to 166 fM only achieved by measuring for 1 -2 hours 11,29,30 . Combining LUNAS with RPA Although the ~1 pM limit of detection of LUNAS is excellent for a direct nucleic acid sensor, the detection of pathogen nucleic acids from diagnostic samples typically requires sensitivity in the attomolar concentration range [19][20][21] .…”