Fig. 5: Detection of microRNA using the mechanism of microRNA as the spacer region in the split sgRNA strategy for Cas12b.

a Schematic representation illustrating how microRNA can replace the original Spacer of split sgRNA, thereby guiding the activity of Cas12b. b Experiments supporting the selective detection of different microRNAs using the split strategy paired with a DNA activator specific to miR-21. Notably, other microRNAs, as well as the precursor of miR-21, do not yield fluorescence. Error bars indicate the mean value +/− SD (n = 3) of biologically independent samples and statistical analysis was conducted using a two-tailed t-test. c Detection of microRNA using commercial paper test strips with various concentrations of miR-21. The photograph shows the test strip results, where the T line indicates the presence of miR-21 and the C line serves as a control. All the experiments were conducted in triplicate and error bars represent mean value +/− SD (n = 3). d Schematic representation of ImageJ quantitative analysis applied to the results shown in (c). Error bars indicate the mean value +/− SD (n = 3) of biologically independent samples, and statistical analysis was conducted using a two-tailed t-test. e Detailed design for evaluating Cas12b response to single-nucleotide mutations using the split strategy. WT refers to the wild-type DNA activator sequence, which is devoid of mutations. The first three bases, TTG, are Cas12b PAM sequence, and the rest 20 bases following the PAM correspond to the Spacer region of Cas12b. The red-highlighted bases indicate mutation sites, with M20 denoting a mutation at the 20th base from the 3’ end of PAM, and M1 corresponding to a mutation at the 1st base from the same end. Single-nucleotide mutations (A-T, T-A, G-C, or C-G) were designed to maintain a constant GC content within the double-stranded DNA activator. f Comparison of fluorescence readings for full-length sgRNA versus split sgRNA (tracrDR+Spacer) when detecting wild-type and single base mutations (M1-20). The y-axis represents the mutation position, correlating with (e), while the x-axis indicates the ratio of fluorescence values obtained from the mutated sequences compared to the wild type. Green bars represent full-length sgRNA, while brown bars represent the tracrDR+Spacer configuration. Asterisks on the right (*,**) signify significant differences between full-length sgRNA and tracrDR+Spacer, with the accompanying numbers indicating the fold change between them. Error bars indicate the mean value +/− SD (n = 3) of biologically independent samples and statistical analysis was conducted using a two-tailed t-test. g Similar to (f), this figure compares fluorescence values of full-length sgRNA and split sgRNA (tracrRNA+crRNA+Spacer) in detecting wild-type and M1-20 single base mutations. Error bars indicate the mean value +/− SD (n = 3) of biologically independent samples, and statistical analysis was conducted using a two-tailed t-test. h The tracrDR matches with DNA activators corresponding to miR-17, 21, 31, and 92a, assessing the levels of these microRNAs in total RNA extracted from colorectal cancer cell lines HCT116 and SW480, as well as non-cancerous intestinal cell line NCM460, within the developed CRISPR-Cas12b system. Different colors represent varying concentrations, with specific values provided in the boxes. i Results of miR-17, 21, 31, and 92a concentration detection in total RNA from the plasma of healthy donors (H.D.-1-5), colorectal cancer patients pre-operation (P.D.-1-5 preO), and post-operation (P.D.-1-5 postO). Different colors denote distinct concentrations, with specific values shown in the boxes. j Comparison of the miR-21 concentration detected in total RNA from the plasma of healthy donors (H.D.-1-5), preoperative colon cancer patients (P.D.-1-5 preO), and postoperative patients (P.D.-1-5 postO) using our current Cas12b split sgRNA approach (tracrDR+miR-21 corresponding DNA activator) versus traditional RT-qPCR methods. The median expression level is depicted by the center line, the interquartile range by the box boundaries, and the whisker extrema represent the maximum and minimum values. Source data are provided as a Source Data file. This Fig. 5a is adapted from Wang, J., Zhang, W., Li, W., Xie, Q., Zang, Z. and Liu, C. Enhancement of CRISPR-Cas12a system through universal circular RNA design. Cell Rep. Methods 5, 101076 (2025)⁶², licensed under CC-BY 4.0 (https://creativecommons.org/licenses/by/4.0/).