Fig. 5: Specific substrate recognition by Ba1Cas12a3 expands the scale of multiplexed RNA detection.
From: RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity
a, Fluorescence production after cleavage of FAM-labelled or quencher-tagged RNA substrates by Ba1Cas12a3, Sm3Cas12a3 and ca23Cas12a3. The area of each circle indicates fluorescence normalized to the highest value for each nuclease, whereas the colour of each circle represents absolute fluorescence production divided by the concentration of nuclease in the reaction. b, Fluorescence production after cleavage of additional FAM-labelled or quencher-tagged RNA substrates by Ba1Cas12a3. c, Fluorescence production after cleavage of FAM-labelled or quencher-tagged RNA substrates specific to Ba1Cas12a3, LwaCas13a or PsmCas13b. d, Schematic of the combined components for multiplexed one-pot detection. In this setup, each fluorophore (FAM, TEX and HEX) can be independently quantified. e, Impact of varying the applied concentration of detected RNAs derived from respiratory syncytial virus (RSV), influenza virus A (IVA) and SARS-CoV-2 (SARS) as part of the one-pot setup. f, Impact of adding human total RNA on one-pot RNA detection. Ratios are based on molarity, assuming an average RNA length of 2 kb in the human total RNA. g, Multiplexed, one-pot RNA detection in the presence of an excess of human total RNA. Dots depict individual measurements of independently prepared reactions, whereas bars and error bars represent the mean ± s.d. of independent measurements (n = 4). Fluorescence measurements depicted as circles or heatmaps represent the average of independent cleavage reactions (n = 3 or 4 in a and b, n = 4 in e and f, n = 6 in g).
