Fig. 1: Development and characterization of the asymmetric cis/trans-cleavage one-pot CRISPR-TB (ActCRISPR-TB) assay. | Nature Communications

Fig. 1: Development and characterization of the asymmetric cis/trans-cleavage one-pot CRISPR-TB (ActCRISPR-TB) assay.

From: Sensitive pathogen DNA detection by a multi-guide RNA Cas12a assay favoring trans- versus cis-cleavage

Fig. 1

a Schematic of Mtb genome IS6110 sites and binding sites for gRNA-0 and six PAM-deficient gRNAs (gRNA-1 to gRNA-6) (Table S1 and Fig. S2). Created in BioRender. Huang, Z. (2025) https://BioRender.com/a99265m. b Relative specificity of the indicated Cas12a/gRNA RNPs for genomic DNA from four Mtb complex (MTBC), seven non-tuberculous mycobacteria (NTM), and three non-mycobacterial species. c Dynamic interplay models of RPA and CRISPR reactions in one-pot RPA-CRISPR assays, established using Cas12a/gRNA RNPs with distinct cis- and trans-cleavage activity profiles. Created in BioRender. Huang, Z. (2025) https://BioRender.com/a99265m. d The cis-cleavage activity (left) and Michaelis−Menten trans-cleavage kinetics (right) of these RNPs, as derived from three experimental and technical replicates, respectively. e Signal kinetics of these one-pot RPA/CRISPR assays detected with a positive control (PC; 1000 copies IS6110 DNA/μL) and a non-templated control (NTC; nuclease-free water). f Schematic depicting the effect of multiple Cas12a/gRNA RNPs to further increase reporter cleavage kinetics. Created in BioRender. Huang, Z. (2025) https://BioRender.com/wtcftat. g, h CRISPR kinetics detected with (g) gRNA-5 or gRNA-5 and gRNA-0, -1, -2, or -4 (1:1 ratio) at constant RNP concentration, or h gRNA-5+gRNA-2, with or without gRNA-0, -1, -3, or -4 (6:1:1 or 3:1 ratio, respectively). One-pot ActCRISPR-TB i kinetics and signal detected with the indicated j IS6110 DNA concentrations and k pathogen genomic DNAs. Data indicate four-parameter logistic (d-left, e, g–i) and Michaelis-Menten (d, right) curve values with 95% confidence intervals (error bars or shaded areas) for representative data, and mean ± standard deviation (SD) values of five technical replicates of the indicated samples (j, k). P values were calculated using a two-sided Wilcoxon signed-rank (e) and Mann-Whitney tests (j, k). Source data are provided as a Source Data file.

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