Dynamic biosensing enables amplifier-collateral-cleavage enhancement for pathogen diagnostic

While CRISPR-Cas system and dynamic DNA nanotechnology have been extensively applied to mainstream biomedical domains, including gene editing, biochemical analysis, and molecular imaging-current approaches remain constrained by limitations in addressing increasingly nuanced and specialized experimental scenarios. Here, we report that CRISPR-CasΦ possesses a unique collateral-cleavage blockade characteristic: CasΦ is unable to recognize the "TTN" sequence in the loop region at the 3' end of stem-loop DNA, resulting in the blockade of collateral-cleavage activity. Leveraging this discovery, we successfully designed and customized two back-end signal amplifiers for biosensing by integrating dynamic DNA sensing studies of CasΦ. Based on these two specialized stem-loop amplifiers, we further developed the Amplifier-collateral-cleavage enhancement (ACE) method, achieving exponential signal amplification. Clinical validation using 112 urine samples demonstrated that ACE exhibits 98.8 % sensitivity and 90 % specificity. These findings highlight the potential of CasΦ dynamic sensing and establish a bridge for future integration of dynamic DNA technology and CRISPR systems.