Fig. 2: High temperature boosted Cas12a trans-cleavage activity results from dsDNA target activation.

a Design of 15 crRNAs targeting the S gene of SARS-CoV-2 with canonical TTTA as PAM. Number these crRNAs from 1-15. b Design of 24 crRNAs targeting the S gene with non-canonical PAMs. Number these crRNAs from 16-39. c Evaluation of the trans-cleavage activity of different PAM-mediated activated LbCas12a at different temperatures. A target final concentration of 100 pM S-gene fragment was used, and the 39 crRNAs mentioned above were used to determine the collateral activity across the temperature range of 37 °C to 57 °C. Values are shown as endpoint fluorescence at 30 min. d Evaluation of the thermal stability of LbCas12a. LbCas12a was pre-incubated at 37 °C, 45 °C, or 53 °C for 30 min, after which the remaining components (crRNA, dsDNA target and reporter) were added for the 45 °C reaction and real-time fluorescence acquisition. e Evaluation of the thermal stability of crRNA. CrRNA was pre-incubated at 37 °C, 45 °C, or 53 °C for 30 min, after which the remaining components (LbCas12a, dsDNA target and reporter) were added for the 45 °C reaction and real-time fluorescence acquisition. f Evaluation of the thermal stability of LbCas12a-crRNA RNP. RNP was pre-incubated at 37 °C, 45 °C, or 53 °C for 30 min, after which the remaining components (dsDNA target and reporter) were added for the 45 °C reaction and real-time fluorescence acquisition. g Evaluation of the thermal stability of dsDNA target activated LbCas12a. LbCas12a-crRNA RNP and target DNA were pre-incubated at 37 °C, 45 °C, or 53 °C for 30 min, after which the reporter was added for the 45 °C reaction and real-time fluorescence acquisition. ① and ② represent the order of addition. Data are represented as mean ± standard error (n = 3 technical replicates). F.I. represents fluorescence intensity. a.u. represents arbitrary units. Source data are provided as a Source Data file.