Fig 1.
Schematic representation of 7SL RNA and Type III CRISPR Cas10 detection system. A. Predicted secondary structure of the T. congolense full 7SL RNA molecule.
The 7SL-sRNA fragment that serves as the diagnostic target is highlighted in red. B: Outline of type III CRISPR-Cas (Cmr)/NucC assay for the detection of RNA. Upon target RNA binding the cyclase (Palm) domain of the Cas10 subunit is activated to produce approximately 1,000 cA3 (3’-5’-cyclic triAMP) molecules per target RNA. The cyclic oligoadenylate in turn activates the nuclease NucC to degrade the dsDNA reporter. Reporters labelled with a fluorophore:quencher pair as shown are monitored by following the development of fluorescence. The reporter can be adapted to enable detection by lateral flow test. Figure created with BioRender.com.
Table 1.
Nucleic acid sequences.
Fig 2.
Effect of crRNA design on assay sensitivity.
Extending the length for the PFS (light blue) at the cost of base-pairing decreases sensitivity 100-fold, from 100 fM (5 nt PFS) to 10 pM (8 nt PFS). The dotted line indicates 1.5-fold change in fluorescence intensity relative to non-target control which is set as the detection threshold. Mean of technical duplicates is shown.
Fig 3.
VmeCmr/NucC assay time for LoD determination.
The development of fluorescence intensity in response to target RNA is measured over time. The fluorescence intensity is then expressed relative to the non-target (negative) control sample. Any change in intensity >1.5-fold (horizontal dotted line) higher than the negative control is taken as positive for the presence of target RNA. Vertical line indicates 120 min as the optimal read time based on experiment length, background signal and sensitivity. The example shown is for the T. brucei 7SL-sRNA-targeting Cmr using synthetic Tbr-7SL-sRNA as the target.
Fig 4.
Limit of detection for T. congolense and T. brucei and discrimination against related species.
A: Synthetic 7SL-sRNA from either species was tested with Cmr[Tco] and Cmr[Tbr] complexes in the Cmr/NucC fluorescent assay. The results give the change in fluorescence intensity relative to the non-target control. The dotted line indicates a 1.5-fold change in fluorescence and demarcates the detection threshold. The mean values with SD from 3 independent experiments are shown. NTC: non-target control. B: Pairwise alignment of the two 7SL-sRNA species used in this study.
Fig 5.
7SL-sRNA detection from in vitro T. congolense (A) and T. brucei (B) cell culture supernatants.
Fold change for Cmr/NucC fluorescence and RT-qPCR assays are shown on the left y-axis. The parasite cell count is shown on the right y-axis. Filled symbols indicate a positive test (above threshold), whereas open symbols indicate a negative test result for the Cmr/ NucC and qPCR assays. The mean and SD from triplicates are shown.
Fig 6.
Detection of Tbr- and Tco-7SL-sRNA using the Cmr/NucC assay with LFT read-out.
A: Schematic representation of the LFT components. The lateral flow strip contains an area for sample application that is followed by deposited gold nanoparticles (GNPs) conjugated to anti FAM antibodies giving a deep purple colour. The sample migrates towards the adsorption pad with capillary force taking the GNPs along. First a line of immobilised streptavidin (control or C line) is traversed, subsequently a line of anti-rabbit antibodies (test or T line). The dsDNA reporter for the LFT is labelled with FAM, the ligand for the GNP conjugate, on one end and with biotin for streptavidin binding at the other end. B: Mechanism of varying test results for the LFT. In the absence of reporter, the GNP conjugate binds to the antibody at the T line. Both the intact reporter and the biotin-labelled degraded reporter after cA3-activated NucC cleavage bind to streptavidin at the C line. However, the GNP conjugate can only bind to the C line when the reporter is intact and labelled with FAM. Any unbound GNP conjugates will then bind to the T line. The reporter concentration must be chosen carefully to obtain meaningful results. C and D: LFT tests were performed on RNA extracted from cultures inoculated with T. congolense (C) or T. brucei (D). The RNA extracts were the same ones used for the assays shown in Fig 5A and 5B, respectively. NTC: non-target control; PC: positive control, 10 pM cognate synthetic 7SL sRNA. The results of each LFT is indicated above the strip with “-“ for a negative test result (no 7SL sRNA detected), and with “+” for a positive test result (7SL sRNA detected). Figure created with BioRender.com.
Fig 7.
Cas10-based detection of 7SL-sRNA during in vivo infection.
RNA was isolated from serum samples derived from three calves experimentally challenged with T. congolense for 63 days. The graphs show the results for each animal from Cmr/NucC fluorescence (circles) and RT-qPCR (diamonds) assays (left y-axis expressed as change in signal relative to negative control), and microscopically determined parasitaemia (bars, right y-axis). Filled symbols indicate a positive test (above threshold), whereas open symbols indicate a negative test result. The results from the LFT with Cmr[Tco] for the same samples are shown on the right with the test result indicated above each strip (+ for positive, - for negative). dpi: days post infection; T: test line; C: control line.