Fig. 5: Atypical gRNA-activated Transcription Halting Alarm (AGATHA) system.

a Schematic showing the design and mechanism of AGATHA sensor. In the box surrounded by the dashed line, the bordered green bar indicates the Broccoli RNA sequence, and the bordered yellow bar indicates the CRISPR target DNA site paired with the target RNA. The bordered dark purple bar indicates PAM, and the bordered light purple bar indicates an anti-Broccoli tail with an inverted repeat sequence of the Broccoli. The two white triangles mark the position where CRISPR/Cas9 cleaves the AGATHA reporter DNA; the borderless lines indicate gRNA molecules. The cartoon below shows the secondary structure of the RNA aptamer predicted by mfold Web Server³⁹. The polygonal star is the fluorogen, which is activated when binding to the Broccoli aptamer. The colors in the RNA structure diagram are consistent to those in the box above, with the pink segment indicating the T7 terminator. b Specificity test of AGATHA. Two different viral RNA fragments (A and B) from the SARS-CoV-2 virus and the corresponding reprogrammed tracrRNAs were used to test the specificity of AGATHA. The T7 in vitro transcription system was used on addition of 5 ng μL⁻¹ AGATHA DNA, 50 nM synthetic CoV-SARS-2 RNA fragment, 50 nM synthetic tracrRNA and 50 nM SpCas9. Data were collected 3 h following incubation. Statistical difference was determined by a two-tailed t test: R1, p < 0.0001, t = 20.57. Error bars, mean values ± s.d. (n = 3). c Schematic showing the assay procedure of AGATHA to detect target coronavirus (SARS-CoV-2) RNA fragments. The reaction was carried out either with (top) or without (bottom) pre-incubation (see METHODS). d Dynamic output responses (left) and mobile phone images (right) of the AGATHA sensor responding to SARS-CoV-2 RNA fragment with (top) or without (bottom) pre-incubation. Phone images were acquired at the end of the reactions (see METHODS). The T7 in vitro transcription system is the same as in b. Error bars, mean values ±s.d. (n = 3). a.u., arbitrary units. p value summary: ****p value < 0.0001, 0.0001 < ***p value < 0.001, 0.001 < **p value < 0.01, 0.01 < *p value < 0.05, p value ≥ 0.05: ns. Source data are provided as a Source Data file.