Fig. 4: Cis-cleavage activities of 120 PAMs of 4 targets.
a, Correlation of one-pot reaction and cis cleavage of 120 PAMs. Black dots represent canonical PAMs. Red dots represent suboptimal PAMs with better performance and blue dots represent suboptimal PAMs with comparable or worse performance than canonical PAMs in one-pot reactions. The unit of one-pot reaction (x axis) is defined as the time to half-maximum fluorescence (min) × an adjusted ratio based on the plateau signal of each PAM. This ratio is the value of the highest plateau fluorescence among 120 PAMs divided by the plateau fluorescence value of each PAM. The 3 suboptimal PAMs out of the 30 min range in the x axis still outperformed their corresponding canonical PAMs. One suboptimal PAM had similar Kcleave value as canonical PAMs, and it performed similarly as canonical PAMs in the one-pot reaction. b, Schematic workflow of amplification and cleavage in one-pot reactions. For substrates containing canonical PAM, cleavage is predominant in the initial stage of the reaction, resulting in excessive consumption of the dsDNA activator. In contrast, as amplification outcompetes cleavage for substrates containing suboptimal PAM, amplicons accumulate to stimulate faster and stronger fluorescence signal production. The up arrows represent the dominant process in the one-pot reactions.
