Low-temperature-tolerant Cas12a enables instrument-free detection of fungal pathogens at mushroom cultivation temperatures.

Reliable molecular diagnosis at the point of care remains challenging owning to its dependence on specialized instrumentation and temperature control. However, clustered regularly interspaced short palindromic repeats (CRISPR) and the associated Cas12a protein have shown promise for point-of-care detection. Therefore, an engineered Cas12a variant from Lachnospiraceae bacterium, namely L.b. Cas12a Ultra, was developed. It exhibited markedly enhanced functional stability and low-temperature tolerance, maintaining robust activity across typical mushroom cultivation temperatures (21 °C-29 °C), compared with wild-type LbaCas12a. By combing this low-temperature-tolerance Cas12a with recombinase-aided amplification (RAA), an instrument-free nucleic acid detection system, termed RAA-CUTE, was established targeting the mushroom cobweb disease pathogen Cladobotryum. This system operates within the typical temperature range for mushroom cultivation. Visualization of RAA-CUTE is achieved using lateral-flow assay strips and fluorescent probes. The method shows high specificity and selectivity in complex sample matrices, with a detection limit of 10 fM and minimal background interference. It also effectively discriminates closely related species through specially designed CRISPR crRNAs, as confirmed in validation assays. This single-tube detection platform provides a sensitive, specific, and field-deployable solution for molecular diagnostics in settings with constrained temperature control and instrumentation, making it a promising tool for on-site crop disease surveillance.