Engineering gRNA for CRISPR biosensing: chemical, structural, and programmable designs for ultrasensitive diagnostics

CRISPR-Cas systems have emerged as powerful biosensing platforms capable of highly sensitive and specific nucleic acid detection. The performance of these assays critically depends on the design of the guide RNA (gRNA), which governs target recognition and activates cis - and trans -cleavage. However, native gRNAs often exhibit limited stability, weak mismatch discrimination, reduced activity in clinical matrices, and inconsistent collateral cleavage, thereby limiting the diagnostic potential of CRISPR sensors. To address these constraints, a wide spectrum of gRNA engineering strategies has been developed, including length change, structural reconfiguration, DNA modified strategies, chemical modifications, mismatch as well as multiple modifications. These modifications substantially enhance sensitivity, specificity, stability, and programmability across Cas12a, Cas13a, and emerging CRISPR effectors. This review systematically summarizes recent advances, compares mechanistic principles and functional outcomes, and highlights future opportunities for rational gRNA engineering to enable amplification-free detection, improve clinical robustness, and drive the development of next-generation CRISPR diagnostics.