Fig. 1

Development and validation of EasyCatch system for specific and sensitive mutation detection. a Schemes of EasyCatch compared with CRISPR detection. b Sequences of D835Y-crRNAs, and FLT3-WT and D835Y gene region. Base G mutates to T in D835Y. c Fluorescence heatmap of different D835Y-crRNA-induced Cas12a reactions detecting 1e10 copies of PCR fragments with different D835Y mutation rates (100, 50, 10%, and 0), Cas12a reactions for 10, 20 and 30 min were recorded. d Specificity assay of D835Y-crRNA2, D835H-crRNA, D835V-crRNA, and D835F-crRNA. Time-course of fluorescence intensity and naked-eye observation after 60 min of Cas12a reaction are shown. e Schematic diagram of MMT-crRNAs-guided Cas12a reaction to identify D835Y/H/V/F mutations from WT background. f Specificity assay of MMT-crRNAs using 1e10 copies of D835Y/H/V/F and WT fragments. Fluorescence intensity after 60 min of Cas12a reaction are shown. IC, inner control. g Time-course analysis of the detection of 1e1 D835Y plasmid templates by RPA combined with MMT-crRNAs induced Cas12a reaction. h, i Sensitivity comparison of EasyCatch and CRISPR detection in detecting 1e6 copies of plasmid templates with gradient D835Y mutation rates. j FGS results of the amplified products in EasyCatch and CRISPR detection. The D835Y mutation rates were quantified using the online tool EditR (https://moriaritylab.shinyapps.io/editr_v10/). k The design and amplification plot of D835Y-probe-mediated qPCR in detecting 1e6 copies of plasmid templates with gradient D835Y mutation rates. l Ct value comparison of different qPCR samples