Fig. 1: Suboptimal PAMs mediated a faster one-pot reaction than canonical PAMs.
a–d, The fluorescence signal of Orf1ab gene spacer 4 and spacer 5 in collateral activity tests (a,b) and one-pot reactions (c,d) at 37 °C. Suboptimal PAMs for Orf1ab spacer 4 (GTTG) and spacer 5 (CTTA) were mutated to canonical PAMs for spacer 4 (TTTG) and spacer 5 (TTTA), respectively. e–h, Summary map of fluorescent kinetics for positions 1–3 point-mutated suboptimal PAMs and three canonical PAMs in collateral activity test (e,f) and the corresponding one-pot reaction of spacer 4 (g) and spacer 5 (h). Time to half-maximum fluorescence was determined. Fluorescence values were determined at 40 and 20 min for collateral activities and one-pot reactions, respectively. The concentrations of dsDNA substrates were 3.5 nM in collateral activity tests and 2,340 fM in one-pot reactions. a,c,e,g, Orf1ab spacer 4. b,d,f,h, Orf1ab spacer 5. Mean ± s.d. of n = 3 technical replicates for e–h. NC represents reactions without substrate in a–d.
