Fig. 1

Specificity and sensitivity of Cas12b-mediated DNA detection. a, b (Upper) Schematics showing mismatched position in the targeting sequence. PAM sequences are colored in red, protospacers are colored in blue, SNPs are colored in pink. (Lower) Characterization of trans-cleavage activity of AaCas12b using ssDNA or dsDNA activator with indicated a single mismatch or b continuous mismatches. Error bars indicate standard errors of the mean (s.e.m.)., n = 3. RFU, relative fluorescence units; PT, perfect target; mPAM, mutated PAM. Two-tailed Student’s t test is used for significance analysis between each mutated sample and the PT sample. c Comparison of the specificity among AaCas12b, PrCas12a, and LbCas12a in dsDNA distinguishability using synthetic HPV16 activator (site 2). Error bars indicate s.e.m., n = 3. d Comparison of trans-cleavage activity and pre-amplification enhanced trans-cleavage activity (CDetection) for AaCas12b using dsDNA activator. AaCas12b is incubated with a sgRNA targeting a synthetic dsDNA 1. Error bars indicate s.e.m., n = 3. RPA, recombinase polymerase amplification. Two-tailed Student’s t test is used for significance analysis between the two samples in each group. e Maximum fluorescence signal obtained from AaCas12b-, PrCas12a-, and LbCas12a-based DNA detection with RPA pre-amplification. Cas12 proteins are incubated with their cognate guide RNA (gRNA) targeting a synthetic HPV16 dsDNA (site 2) mixed with background genome. Error bars indicate s.e.m., n = 3. Two-tailed Student’s t test is used for significance analysis among each other in the group. f Fluorescence timecourses obtained from AaCas12b- and LbCas12a-based DNA detection with RPA pre-amplification. Cas12 is incubated with a cognate gRNA targeting a synthetic HPV16 dsDNA (site 2) diluted in human plasma with a final concentration of 10⁻¹⁸ M. Error bars indicate s.e.m., n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance