An allosteric key strand controlled adaptable CRISPR/Cas12a biosensing platform for point-of-care testing of multiple...

Currently, the CRISPR/Cas12a based sensor has become a powerful tool for gene editing and molecular diagnostics. However, most CRISPR/Cas12a sensors are primarily limited to the detection of a single target type, due to their strict dependence on the specific recognition of the PAM sequence within a precisely designed double-stranded DNA (dsDNA) and crRNA for cleavage activity regulation. Herein, we designed an allosteric key strand (KS) controlled CRISPR/Cas12a biosensor via toehold-based strand displacement reaction (TSDR). By simply reconfiguring KS into different conformations with functional nucleic acid structures, this sensor could selectively respond to various target molecules from nucleic acids to non-nucleic acid molecules without changing the sequence of crRNA and targeted PAM-dsDNA. The trans-cleavage activity of CRISPR/Cas12a could be triggered through leveraging proximity-based TSDR in response to target binding. The proposed sensor achieved sensitive and specific detection of various targets, including nucleic acids (HPV-16), small molecules (kanamycin), and enzymes (uracil-DNA glycosylase). Furthermore, by integrating lateral flow assay technology, this CRISPR/Cas12a-based system enabled point-of-care testing (POCT) for the detection of multiple target types. This approach can overcome the sequence-specific limitations, thereby improving the versatility of CRISPR/Cas12a sensors for extending more target types detection. We anticipate this innovative technology will serve as a flexible and accessible sensing platform, facilitating rapid diagnosis in the field of POCT and enabling its broader application across diverse biotechnological domains.