Split crRNA Precisely Assisted Cas12a Expansion Strategy for Simultaneous, Discriminative, and Low-Threshold Determination of Two miRNAs Associated with Multiple Sclerosis

Multiple sclerosis (MS) can proceed into secondary progressive MS accompanied by persistent neurological deterioration; therefore, accurate diagnosis of MS is of vital significance. Irregularities of microRNAs (miRNAs) expression have been observed in MS, so miRNAs have been evaluated as novel biomarkers and therapeutic targets. Herein, a new strategy named <u>s</u>plit crRNA <u>p</u>recisely <u>a</u>ssisted <u>C</u>as12a <u>e</u>xpansion (SPACE) was developed for simultaneous, discriminative, and low-threshold determination of two MS-related miRNAs: miRNA-155 and miRNA-326. On the one hand, owing to the property that split crRNA could activate Cas12a, miRNAs were designed as the spacers of crRNA to combine with scaffold. These integrated crRNAs then recognized the activators, activating Cas12a and enabling RNA target identification. On the other hand, the SPACE strategy dexterously integrated the activator with reporter probe, and utilized Cas12a's <i>cis</i>-cleavage to achieve simultaneous detection and differential signal output for miRNA-155 and miRNA-326. Moreover, <i>trans</i>-cleavage with ultra-high efficiency was assembled in the SPACE strategy to achieve sensitive quantification of total miRNAs in blood samples at low thresholds. Overall, the diversified and integrated design of the SPACE strategy enabled simultaneous, discriminative, and low-threshold detection of dual MS-related miRNAs in one pot and one step, providing a reliable and accurate Cas12a detection tool for clinical low-threshold diagnosis.