Fig. 2: PAM-free nucleic acid barcoding amplification leads to a CRISPR induced cis-cleavage output.

a Schematic of the generation of a composite DNA encoding crRNA (DNAcrRNA) upon target nucleic acid amplification using P1 and P2 primers. Following transcription (Tx), the functionalized CRISPR output product can be cleaved and loaded into Cas12a. The DNAcrRNA serves as an encoded barcode specifying which nucleic acid was amplified. The correct combination of the crRNA and molecular beacon (containing a PAM+ protospacer) generates a fluorescent output. b RT-HDA was performed using synthetic target RNA (Supplementary Data 1, line 93) at 5 pM or H₂O (neg). The barcoded product was then transcribed and combined with FnCas12a and the reporter dsDNA molecular beacon. TAMRA fluorescence (543/584 nm) was monitored in real time at 37 °C in a plate reader. c Using the data from b, the initial rate of fluorescence increase within 1 h (60 min) was plotted. d HDA was performed using synthetic target DNA at 5 pM or H₂O (neg). The barcoded product was then transcribed and combined with FnCas12a and the reporter dsDNA molecular beacon. TAMRA fluorescence was monitored in real time at 37 °C in a plate reader. e Using the data from d, the initial rate of fluorescence increase within 1 h (60 min) was plotted. Error bars: mean ± SD. Experiments displayed are representative of independent biological triplicates (n = 3). Source data are provided as a Source Data file.