Fig. 2

Wild-type TsCas12a performs optimally in RPA reactions across a broad temperature window. (a). Schematic of one-pot RPA-Cas12a reaction. (b). RPA-Cas12a reactions were conducted with LbCas12a at varied temperatures. Symbols represent mean (± SD) fluorescence values for replicates (n = 3) in the absence (open symbols) or presence (filled symbols) of HPV-16 plasmid. Control reactions carried out in the absence of target are obscured. (c) RPA-Cas12a reactions were conducted with different Cas12a orthologs at varied temperatures in the absence or presence of HPV-16 plasmid, and fluorescence signals were recorded (Supplementary Fig. 2). Heat-map represents average values observed at 60 min in response to target. (d) RPA-Cas12a reactions were conducted with TsCas12a as in (b).(e, f). RPA-Cas reactions were conducted with TsCas12a at 25 °C in the absence (open symbols) or presence (filled symbols) of plasmids encoding genomes of various HPV types. Symbols (e) represent mean (± SD) fluorescence values of replicates (n = 3), with bars (f) representing those recorded at 60 min. (g). RPA-Cas reactions were conducted with TsCas12a at 25 °C in the absence or presence of 3.0 ng of human genomic DNA and HPV-16 plasmid. Symbols represent mean (± SD) fluorescence values of replicates (n = 3). (h). RPA-Cas12a reactions were conducted with a 1:1 mixture of Lb and TsCas12a as in (b). (i). Heat-map represents average values observed at 30 min for RPA-Cas reactions using Lb and TsCas12a, alone or combined, in response to target from panels (b), (d), and (h).