Fig. 1: The trans-cleavage activity of LbCas12a with modified crRNA via fluorescence-quencher-based reporter assay with TA rich fluorophore-quencher systems tested.

For other fluorophore-quencher systems, see Supplementary Figs. 1–4. a Schematic diagram of cleavage of Cas12a with wild-type and modified crRNAs. The crRNA is extended on either the 3′- or 5′-ends with ssDNA, ssRNA, or phosphorothioate ssDNA. b A representation of a fluorescence-quencher-based trans-cleavage reporter assay image taken by GE Amersham Typhoon. c, d, and e 3′-end ssDNA, ssRNA, and phosphorothioate ssDNA extensions of crRNA, respectively. f, g, and h 5′-end ssDNA, ssRNA, and phosphorothioate ssDNA extensions of crRNA, respectively. The fold in fluorescence was normalized by taking the ratio of background-corrected fluorescence signal of a sample with the activator to the corresponding sample without activator. For c–h, error bars represent mean ± SEM, where n = 6 replicates (three technical replicates examined over two independent experiments). Statistical analysis was performed using a two-way ANOVA test with Dunnett’s multiple comparison test, where ns = not significant with p > 0.05, and the asterisks (*, **, ***, ****) denote significant differences with p values listed above. Source data are available in the Source Data file.