Simply Engineered crRNA with CRISPR/Cas12a System Enables Wide-Scope Nucleic Acid Biomarker Analysis.

CRISPR/Cas12a systems have emerged as versatile tools for molecular diagnostics, but directly detecting RNA and identifying specific DNA point mutations remain challenging. Herein, we report a simple engineering approach with a split site in the spacer sequence, enabling activation of CRISPR/Cas12a (LbCas12a) for trans-cleavage with similar efficiency to wild-type crRNA. The engineered crRNA facilitated RNA target recognition by replacing the 3'-end with RNA fragments, enhancing point mutation specificity for ssDNA targets. Based on this, we achieved amplification-free detection of microRNAs and DNA point mutations with high sensitivity and specificity. For clinical sample validation, we constructed reverse fluorescence-enhanced lateral flow test strips (rLFTS), which achieved femtomole-level detection. Moreover, the engineered crRNA-based CRISPR/Cas12a system also effectively recognized tumor cells via intracellular and in vivo imaging of miRNA-21. In conclusion, this engineered crRNA platform enhances CRISPR/Cas12a-based nucleic acid detection, promoting its wide application in molecular diagnostics and bioimaging.