A DNAzyme-activated engineered crRNA enables CRISPR-Cas12a biosensing of ALP and S. aureus

CRISPR-Cas12a biosensing is hindered by insufficient sensitivity that necessitates pre-amplification, as well as the limited ability to directly detect non-nucleic-acid targets. Herein, we developed a CRISPR-Cas12a biosensor centered on a DNAzyme-activated engineered crRNA (e-crRNA), enabling Mn²⁺-responsive control over its trans-cleavage activity for nucleic-acid-amplification-free detection. Upon Mn²⁺-dependent activation, the DNAzyme cleaved the e-crRNA to liberate a full-sized crRNA₁ and a split crRNA₂. The combined crRNA₁/crRNA₂ system enhanced Cas12a trans-cleavage activity, compensating for the activity loss associated with crRNA engineering. Rational design of the split site further improved the signal-to-noise ratio. Moreover, by gating the system activation on Mn²⁺, the non-specific activation effects caused by spurious DNA hybridization were mitigated. When coupled with a Mn²⁺-responsive signal conversion strategy, this platform achieved sensitive detection of alkaline phosphatase with the LOD of 0.008 U/L and Staphylococcus aureus with the LOD of 29.76 CFU/mL. This study showcased the innovative use of Mn²⁺-dependent DNAzyme to efficiently activate CRISPR-Cas12a system, establishing a powerful platform for biosensing.