CRISPR RNA (crRNA) is the key guide molecule in the CRISPR-Cas12a system, directing the Cas protein to recognize target sequences. It consists of a repeat-derived stem loop that binds Cas12a and helps stabilize the ribonucleoprotein complex, and a spacer region that base-pairs with the target and determines recognition specificity. Recently, multiple studies have shown that crRNAs can be split and reassembled in vitro in diverse ways. These split-and-reconfigured strategies have enabled detection schemes that are more flexible than full-length crRNAs, cover a broader range of targets, and achieve higher signal-to-background ratios. Here, we focus on split crRNA strategies for CRISPR-Cas12a and systematically summarize existing split crRNA-based detection platforms. We outline their design principles, reaction mechanisms, and performance features, and we synthesize how these approaches improve key metrics-including target scope, sensitivity, specificity, and controllability. Finally, we discuss the major advantages and current limitations of split crRNA strategies and highlight directions for further design optimization and translational applications. Schematic overview of split crRNA strategies for enhancing CRISPR-Dx performance. These advances are mainly reflected in four aspects: broadening the target range, thereby enabling Cas12a to be applied to the detection of short RNAs, structured RNAs, and certain non-nucleic acid targets; improving sensitivity by enhancing detection signals through reassembly-dependent activation, cascade amplification, or auxiliary activation strategies; increasing specificity by strengthening the discrimination of single-nucleotide differences through stepwise recognition and conditional assembly; and enhancing controllability by achieving on-demand activation of Cas12a activity via light, enzymes, small molecules, or proximity effects.
Guo et al. (Sun,) studied this question.