Fig. 2: In vitro characterization of crRNA mutants.

a The binding affinity of RNPs for dsDNA. dCas12a-crRNA RNPs (0, 30, 60, 90, 120, and 150) were incubated with 5 nM dsDNA at 37 °C for 15 min, and EMSA were performed to determine the bound and unbound dsDNA. b Fluorescent characterization of cis-cleavage. The FAM- and BHQ1-labeled dsDNA probe was cleaved by Cas12a-crRNA RNP to generate fluorescence. Shaded areas around the curves represent the standard deviation (SD) of three independent experiments. c Fluorescent characterization of trans-cleavage at various target dsDNA substrate (10 nM, 1 pM, 10 pM), with 8 C ssDNA probe cleaved to generate fluorescence. Fluorescence intensity at 40 min with background subtracted is shown. d Fluorescence signals from trans-cleavage of RNPs activated by target dsDNA containing double-point mutations. The upper panel shows the sequence of the mutated targets. The lower panel presents the fluorescence signal at 40 min for each crRNA, normalized to the perfect match signal after background subtraction. MM indicates mismatches. Data are shown as mean ± s.d. for n  =  3 biologically independent samples. Source data are provided as a Source Data file.