Fig. 1: Evaluation of different Cas12a-gRNA combinations at room temperature (24 °C).

a Schematic of a fluorescence trans-cleavage assay. Here, the reporter comprises a fluorophore linked to a quencher by a short piece of ssDNA. The gRNA is programmed to recognize a particular locus of the SARS-CoV-2 genome. In the absence of the virus, the reporter molecule is intact and thus no fluorescence is observed. However, when the virus is present, the Cas12a RNP will bind to and cleave its programmed target, become hyperactivated, and cut the linker between the fluorophore and quencher, thereby generating a fluorescence signal. b Organization of the SARS-CoV-2 genome. Genes encoding structural proteins are indicated by green boxes, while genes encoding accessory proteins are indicated by cyan boxes. Although ORF10 is annotated in the genome, there is currently no evidence of its expression⁷¹. The locations of the new gRNAs are shown by pink bars below the genes, while the N-Mam locus is shown by a red bar. c Fluorescence measurements using a microplate reader after 30 min of cleavage reaction. 1E11 copies of the relevant DNA target were present in a 50 μl reaction. All readings were normalized to the no template control (NTC) at the start of the experiment. The N1 gRNA gave an unexpected result, whereby it triggered the collateral activity of AsCas12a and its variants without a template. Data represent mean ± s.e.m. (n = 3 [N-Mam, O1, O2, S2, S3], 4 [S1], or 6 [N1] biological replicates). d Sequences of perfect matched (PM) or mismatched (MM) spacers targeting the N-Mam locus. Each mismatched position is indicated by a bold red letter. e Heatmap showing the tolerance of various Cas12a enzymes to mismatched N-Mam gRNAs. The fluorescence readings are scaled between 0 and 1, where 1 is the highest measurement obtained and 0 is the background signal for NTC at the start of the experiment. f Sequences of perfect matched (PM) or mismatched (MM) spacers targeting the S2 locus. Each mismatched position is indicated by a bold red letter. g Heatmap showing the tolerance of various Cas12a enzymes to mismatched S2 gRNAs. The fluorescence readings are scaled between 0 and 1, where 1 is the highest measurement obtained and 0 is the background signal for NTC at the start of the experiment. Source data are available in the Source Data file.