Fig. 3: Characterization of ENHANCE with various crRNA modifications and different Cas12a systems.

a Comparison of trans-cleavage activity between precursor crRNA (pre-crRNA) and mature crRNA (tru-crRNA, where the first Uracil on the 5′-end of the crRNA is cleaved by LbCas12a in the absence of the activator). b Comparison of trans-cleavage activity between AT-rich extensions and GC-rich 7-nt DNA 3′-end extensions on the crRNA + 3′DNA7. For a, b), error bars denote mean ± SD, where n = 3 technical replicates. c Trans-cleavage activity of LbCas12a with non-fully phosphorothioate (PS) modified crRNA targeting GFP fragment. Sequence representation of 6 non-fully PS extension on the 3′-end of crGFP ranging from 1 to 6 PS. The asterisk symbol (*) signifies the phosphorothioated nucleotide. The graph below the sequence representation shows fold change of the LbCas12a fluorescence-based reporter assay with the activator normalized to the corresponding samples without the activator at t = 20 min. d kinetics of the LbCas12a fluorescence-based reporter assay in c, e Trans-cleavage activity of different variants of Cas12a. The prefix Lb, As, and Fn stand for Lachnospiraceae bacterium, Acidaminococcus, and Francisella novicida, respectively. For c, d, and e, n = 6 replicates (three technical replicates examined over two independent experiments), where error bars in e represent mean ± SEM. Source data are available in the Source Data file.