We present a robust 'splice-at-will' CRISPR RNA (crRNA) engineering mechanism that overcomes the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in directly detecting ultrashort RNAs. In this strategy, an intact Cas12a crRNA can be split from almost any site of the spacer region to obtain a truncated crRNA (tcrRNA) that cannot activate Cas12a even after binding an auxiliary DNA activator. While splicing tcrRNAs with a moiety of ultrashort RNA, the formed combination can work together to activate Cas12a efficiently, enabling 'splice-at-will' crRNA engineering. Importantly, the 'splice-at-will' crRNA exhibits almost the same trans-cleavage activation efficiency as that of a conventional intact crRNA. Therefore, by rationally designing a DNA auxiliary activator with a conserved tcrRNA-complementary sequence and an arbitrary short RNA-of-interest recognition domain, a general sensing system is established that directly utilizes traditional DNA-activated Cas12a to detect ultrashort RNAs. This 'splice-at-will' crRNA engineering strategy could faithfully detect ultrashort RNA sequences as short as 6-8 nt, which cannot be achieved by conventional Cas12a and Cas13a systems. Additionally, through flexible splicing site design, our method can precisely distinguish single-base differences in microRNA and other short RNA sequences. This work has significantly expanded the Cas12a-based diagnostic toolbox and opened new avenues for ultrashort RNA detection.
© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.