An aptamer-regulated CRISPR/Cas12a system based on split crRNA for theophylline and tobramycin detection.

Most existing CRISPR/Cas12a methods for small molecules detection employ DNA aptamers as an intermediary, while RNA aptamers remain underexplored. The conventional CRISPR/Cas12 system offers limited programmability due to its partially conserved crRNA sequence. In this study, we developed an aptamer-regulated CRISPR/Cas12a system based on split crRNA. We divided the full crRNA into a crRNA handle segment (RH) and a crRNA spacer segment that is identical to the RNA aptamer sequence of target (RS-Apt). RS-Apt was endowed with dual functions: activating the activity of Cas12a and binding to target small molecules, ingeniously linking non-nucleic acid targets with the CRISPR system. The strong affinity between the target and RS-Apt reduces the amount of RS-Apt available to participate in the CRISPR system, resulting in a decrease in the fluorescence signal of the system. Within 1.5 h, the system for theophylline (TP) exhibited a linear range from 50 nM to 200 µM and a detection limit (LOD) of 2 nM, while the system for tobramycin (TOB) showed a linear range of 200 nM to 100 µM with an LOD of 63 nM. Notably, visual semi-quantitative detection of TP and TOB was achieved via lateral flow strips. Moreover, the system achieved satisfactory recovery and visual detection of TP using lateral flow strips in TP-spiked serum samples. This system can detect different targets simply by changing the sequence of the RS-Apt and activator, without the need for complex sequence design of the chains or large-scale instruments, providing a new strategy for CRISPR/Cas12a in the detection of small molecules.