Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters

Abstract

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

Signal Amplification by the trans-Cleavage Activity of CRISPR-Cas Systems: Kinetics and Performance

“…When the substrate concentration is not much higher than that of the active Cas enzyme, the reaction is considered a pseudo-first-order reaction, and the reaction rate is k obs multiplied by the substrate concentration ([S]). k obs can be calculated using nonlinear regression of the fluorescence–time curve (Figure and eq 5). , A measurement of k obs requires only one substrate concentration. Therefore, the apparent cleavage rate ( k obs ) is useful for large-scale kinetics comparisons of different enzymes under different conditions.…”

“…k obs can be calculated using nonlinear regression of the fluorescence–time curve ( Figure 3 and eq 5). 48 , 51 A measurement of k obs requires only one substrate concentration. Therefore, the apparent cleavage rate ( k obs ) is useful for large-scale kinetics comparisons of different enzymes under different conditions.…”

Amplification-free CRISPR/Cas detection technology: challenges, strategies, and perspectives

“…Rossetti et al revealed that hairpin DNA reporters generate a faster and enhanced fluorescence signal change compared to linear ssDNA reporters, performing with better sensitivity (10 pM, 10-fold improvement) in the detection of SARS-CoV-2. 50 The kinetic analysis also confirmed better affinity between hairprin reporters and Cas12a, resulting in an ∼5-fold improvement of catalytic efficiency parameter ( k cat / K M ) and a faster trans -cleavage rate. Although the sensitivity failed to meet the estimated LOD requirement of 100 copies per μL for sensitive COVID-19 screening, 51 this work evolutionarily provided a better understanding of Cas12a nuclease activity and could be utilized to integrate with other improved amplification-free CRISPR/Cas-based detection methods.…”

Research progress of CRISPR-based biosensors and bioassays for molecular diagnosis

“…Still, the increase is not significant after the peak of 8 is reached, and the synthesis of excessively long ssDNA also leads to an additional cost of detection (Lv H. et al, 2021). In a recent study, researchers found enhanced trans cleavage activity and significantly improved signal intensity by linking the fluorophore to the quencher with hairpin DNA (Rossetti et al, 2022). These studies set the first step toward continuous improvement of the reaction system in the future.…”