CRISPR/Cas14 provides a promising platform in facile and versatile aptasensing with improved sensitivity.

CRISPR is reshaping biosensing technology due to its programmability, sensitivity, and specificity. Most current CRISPR-based biosensors are developed based on Cas12 and Cas13, while the biosensing potentials of the newly discovered Cas14 have not been fully elucidated yet. Herein, a fluorometric biosensor named HARRY (highly sensitive aptamer-regulated Cas14 R-loop for bioanalysis) was developed. The diblock ssDNA is designed to contain the activator sequence of Cas14 and the aptamer sequence of specific targets. In the absence of targets, the ssDNA activates Cas14a, then the Cas14a trans-cleavages the fluorescent reporter, causing fluorescence enhancement. In the presence of the targets, ssDNA-target assembly is formed via aptamer interaction, resulting in the inhibition of Cas14a activation. HARRY can detect ATP, Cd, histamine, aflatoxin B1, and thrombin with detection limits at the low-nanomolar level, which shows improvement compared with Cas12a-based aptasensors in sensitivity and versatility. We reasoned that the improvement is derived from the ssDNA specificity of Cas14a and found that the detection limit of HARRY is correlated to the binding affinities of aptamers. This study unlocks the potential of Cas14a in versatile aptasensing, which may inspire the development of CRISPR-based biosensors from the Cas14a branch.