Fig. 2: Activity and mismatch tolerance of enAsCas12a with various S-gene-targeting gRNAs. | Nature Communications

Fig. 2: Activity and mismatch tolerance of enAsCas12a with various S-gene-targeting gRNAs.

From: An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing

Fig. 2

a Fluorescence measurements for a single S2 gRNA complexed with various Cas12a nucleases after 30 min of trans-cleavage reaction at 37 °C. Compared to the earlier results obtained at 24 °C, there was still no cross-reactivity for SARS-CoV or MERS-CoV at the higher temperature, but the fluorescence signal for SARS-CoV-2 was approximately twice as high. Data represent mean ± s.e.m. (n = 3 biological replicates). b Heatmap showing the tolerance of various Cas12a enzymes to mismatches at the S2 target site when the trans-cleavage assay was performed at 37 °C. 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. c Fluorescence measurements for enAsCas12a complexed with different gRNAs targeting the S-gene of SARS-CoV-2 after 30 min of cleavage reaction at 37 °C. 1E11 copies of DNA were present in a 50 μl reaction. Two of the gRNAs (S4 and S8) triggered the collateral activity of enAsCas12a without a template. Data represent mean ± s.e.m. (n = 4 [S9], 5 [S4, S5, S6, S7, S11, S14], 6 [S8, S10, S12, S13], or 7 [S15] biological replicates). d Buffering the collateral activity of enAsCas12a against SNVs with a second gRNA. Fluorescence measurements here were taken after 30 min of cleavage reaction at 37 °C. The S6 gRNA was used together with either the perfect matched (PM) or a mismatched (MM10) S2 gRNA in the absence or presence of 0.1 M glycine. Data represent mean ± s.e.m. (n = 3 [with glycine] or 4 [no glycine] biological replicates). (n.s. not significant, P > 0.2; two-sided Student’s t-test). e Analytical limit of detection (LoD) for enAsCas12a complexed with both the S6 gRNA and either the perfect matched (PM) or a mismatched (MM10) S2 gRNA. Different copies of SARS-CoV-2 RNA fragments were used as input to RT-LAMP, which was performed at 65 °C for 15 min using an initial set of LAMP primers (0.2 µM of each displacement primer, 1.6 µM of each internal primer, and 0.8 µM of each loop primer). The Cas detection reaction was then carried out at 37 °C, with the fluorescence measurements here taken after 10 min. Data represent mean ± s.e.m. (n = 6 [2E6] or 7 [other copy numbers] biological replicates). Source data are available in the Source Data file.

Back to article page