A rapid and straightforward detection of the novel obesity gene LGR4 A750T point mutation by the single-tube Cas12a system with split crRNA.

With the increasing number of obesity cases globally, immediate screening for obesity-related mutations has become increasingly important. LGR4 is a novel obesity gene. Here, we report a dual-mode detection strategy for the LGR4 p.A750T genetic variant by integrating asymmetric recombinase polymerase amplification (aRPA) with a split CRISPR-Cas12a system. Through isothermal aRPA amplification, the system directly generates abundant single-stranded DNA products that activate the split Cas12a complex, enabling ultra-sensitive and PAM-independent detection without spatial constraints on mutation sites. By employing a split crRNA design rather than conventional CRISPR-Cas12a, the method demonstrates exceptional specificity in distinguishing DNA point mutations. The assay achieves a detection limit of 13.5 fM, exhibits excellent linearity from 100 fM to 1 nM, and completes analysis within 30 minutes. Furthermore, when coupled with lateral flow test strip visualization, the system eliminates the need for sophisticated instrumentation and enables rapid single-tube amplification for interpretation of results. Based on the high mutation discrimination capability of the SCas12a system, the method achieves highly sensitive and specific LGR4 point mutation detection.