Fundamental limits of amplification-free CRISPR-Cas12 and Cas13 diagnostics

Abstract

Interest in CRISPR diagnostics continues to increase. CRISPR-Cas12 and Cas13 based detection are particularly interesting as they enable highly specific detection of nucleic acids. The fundamental sensitivity limits of Cas12 and Cas13 enzymes are governed by their kinetic rates and are critical to develop amplification-free assays. However, these kinetic rates remain poorly understood and their reporting has been inconsistent. We here measure kinetic parameters for several enzymes (LbCas12a, AsCas12a, AapCas12b, LwaCas13a and LbuCas13a) and evaluate their limits of detection (LoD) for amplification-free target detection. Collectively, we here present quantitation of enzyme kinetics for 14 gRNAs and nucleic acid targets for a total of 50 sets of kinetic rate parameters and 25 LoDs. Importantly, we also validate the self-consistency our measurements by comparing trends and limiting behaviors with a Michaelis-Menten, trans-cleavage reaction kinetics model. Our measurements reveal that activated Cas12 and Cas13 enzymes exhibit typical trans-cleavage catalytic efficiencies between order 105 and 106 M-1 s-1. Moreover, for assays that use fluorescent reporter molecules (ssDNA and ssRNA) for target detection, we find most CRISPR enzymes have an amplification-free LoD in the picomolar range. We find also that successful detection of target requires cleavage (by activated CRISPR enzyme) of at least ~0.1% of the fluorescent reporter molecules. This fraction of cleaved reporters is required to differentiate signal from background, and we hypothesize that this fraction is largely independent of the detection method (i.e., endpoint vs reaction velocity). Our results provide a map of the feasible application range and highlight areas of improvement for the emerging field of CRISPR diagnostics.

Cas12a2 elicits abortive infection via RNA-triggered destruction of double-stranded DNA

“…Collectively these results suggest that the dsDNase activity of Cas12a2 is instrumental in triggering the Abi phenotype. Additionally, as a proof-of-principle demonstration, we show that Cas12a2 can detect RNA at a sensitivity comparable to that of RNA-targeting Cas13a nuclease at various temperatures 18 .…”

“…Cas12a2 holds substantial potential for CRISPR technologies. As a proof-of-principle demonstration, we showed that SuCas12a2 can be repurposed for RNA detection with a limit of detection comparable to existing single-effector based tools 18 . Beyond the ability to detect RNA, we envision a variety of SuCas12a2 applications that expand and enhance the CRISPR-based tool kit.…”

“…*: p < 0.05. **: p < 0.005 ( g ) Unamplified limit of detection (LOD) for several Cas detectors 18 compared to Cas12a2 determined by the velocity detection method. ( h ) Proposed model for promiscuous RNA targeting and collateral DNA degradation by SuCas12a2 and its outcome on cell growth.…”

“…Standard error of the linear fit was used as a proxy for standard deviation, and the limit of detection was calculated as 3 x Standard Error of the water background as in (ref. 18 ). Limit of detection was estimated by determining where the plot of V O vs. [Target RNA] crosses the detection threshold.…”

Detection and discrimination of single nucleotide polymorphisms by quantification of CRISPR-Cas catalytic efficiency

“…A fluorescence calibration curve was then used to convert fluorescence values (in AU) to molar concentrations (in nM). 19,20 To this end, two nonlinear fits were performed on calibration data, one each for cleaved and uncleaved reporters ( Fig. S1 ).…”