Fig. 3. Cascade signal amplification of nucleic acid detection by competitive crRNA.

a Cas12a, full-sized crRNA, and split crRNA were incubated with different combinations of Cy5-full-T and FAM-split-T for 0, 5, 15, and 30 min. More diverse control conditions were shown in Supplementary Figure 1. This experiment was performed twice. M = marker, [Cas12a] = 100 nM, [full-sized crRNA] = 40 nM, [split crRNA] = 10 nM, [Cy5-full-T] = 100 nM, and [FAM-split-T] = 50 nM. b Cas12a, Cy5-full-sized crRNA, and FAM-split crRNA were incubated with different combinations of full-T and split-T for 0 and 30 min. The 5’-Cy5-full-sized crRNA is trimmed at the 5’-end by LbCas12a (the first Cy5-conjugated-Uracil is cleaved).¹⁷ More diverse control conditions were shown in Supplementary Figure 2. This experiment was performed twice. M = marker, [Cas12a] = 200 nM, [Cy5-full-sized crRNA, FAM-split crRNA handle, and split crRNA spacer] = 100 nM, [full-T] = 100 nM, and [split-T] = 50 nM. c Electrophoretic mobility shift assay. The split crRNA binding to Cas12a is inhibited by the full-sized crRNA in the absence of the full-T (left) or in the presence of non-target (middle). The split crRNA can replace the full-sized crRNA-DNA hybrid or the cleaved R-loop in Cas12a after the full-sized crRNA/Cas12a is activated by the full-T (right). This experiment was performed twice. [LbCas12a] = 250 nM, [Cy5-full-sized crRNA] = 200 nM, [Full-T] = 100 nM, and [non-target] = 100 nM. Gel was measured by the Cy5 and Alexa488 filters, respectively, and then merged. The red band shows the Cy5 signal, and the green band shows the FAM/SYBR gold signal. Source data are provided as a Source Data file.