An “Off+Off=On” Amplification-Free Strategy Leveraging Threshold Concentration for Enhanced Detection Sensitivity in Crispr-Cas Biosensing Systems

Abstract

Pre-amplification-free CRISPR/Cas diagnostic biosensors are a research hotspot due to their speed, simplicity and low-cost advantages. Here, we proposed an “OFF + OFF = ON” strategy based on Cas12a inspired by threshold concentration, which enables sensitive and rapid nucleic acid diagnostics without pre-amplification. First, anassisted nucleic acid (AN) with a sequence identical to the target nucleic acid (TN) is added to the detection system at a threshold concentration, maintaining the trans-cleavage activity of Cas12a remains undetectable and the signal in an “OFF” state, which transitions to “ON” when this concentration is exceeded. Similarly, when the TN concentration falls below the threshold concentration, it maintains another “OFF” state. However, when the combined concentrations of AN and TN exceed the threshold concentration, the trans-cleavage activity of CRISPR/Cas12a is activated and the system transitions to an “ON” state, thereby triggering the detectable activation of the biosensor. The strategy can significantly improve the sensitivity for bacteria or nucleic acid detection by two orders of magnitude compared with a direct Cas12a detection system, providing a new pre-amplification-free pathway for developing Cas-based sensors with high sensitivity.

Keywords: CRISPR-Cas12a, Staphylococcus aureus, mecA, biosensor