Biosensing bacterial 16S rDNA by microchip electrophoresis combined with a CRISPR system based on real-time crRNA/Cas12a formation.

The accurate, simple and sensitive detection of bacterial infections at the early stage is highly valuable in preventing the spread of disease. Recently, CRISPR-Cas12a enzyme-derived nucleic acid detection methods have emerged along with the discovery of the indiscriminate single-stranded DNA (ssDNA) cleavage activity of Cas12a. These nucleic acid detection methods are made effective and sensitive by combining them with isothermal amplification technologies. However, most of the proposed CRISPR-Cas12a strategies involve Cas-crRNA complexes in the preassembled mode, which result in inevitable nonspecific background signals. Besides, the signal ssDNA used in these strategies needs tedious pre-labeling of the signal molecules. Herein, a post-assembly CRISPR-Cas12a method has been proposed based on target-induced transcription amplification and real-time crRNA generation for bacterial 16S rDNA biosensing. This strategy is label-free through the combination of microchip electrophoresis (MCE) detection. In addition, this method eliminates the need for a protospacer adjacent motif (PAM) on the target sequences, and has the potential to be an effective and simple method for nucleic acid detection and infectious disease diagnosis.