Fig. 5: A novel type III CRISPR-Cas tool for the sensitive detection of nucleic acids. | Nature Communications

Fig. 5: A novel type III CRISPR-Cas tool for the sensitive detection of nucleic acids.

From: SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation

Fig. 5

a Denaturing PAGE resulting from activity assays performed with the CARF protein TTHB144 and a 5′ Cy5-labeled reporter RNA. Activation of TTHB144 due to cOAs produced by the reconstituted TtCmr-46 complex was monitored by offering fully complementary (T) target RNAs, or target RNA with mismatches in segments one (S1), four (S4) or by a single mismatch in the CAR (C1). A fully non-target RNA (NT) was used as a control. Results of the TTHB144 cleavage assay is representative of results obtained from three replicates (Supplementary Fig. S6). b Limit of detection (LOD) assay using reconstituted Cmr-46 on a synthetic SARS-CoV-2 E-gene and a fluorophore-quencher reporter RNA masking construct measured over time. A non-target RNA (NT) was used as a control. Data was obtained from three replicates. c Schematic overview of the 2-step reaction setup consisting out of (1) a (RT)-LAMP based pre-amplification step and (2) a T7-based in vitro transcription and type III CRISPR detection step. ‘F-Q’ represents the fluorophore-quencher reporter RNA. d Limit of detection assay using reconstituted Cmr-46 in the 2-step setup (depicted in panel c), with a SARS-CoV-2 synthetic full RNA genome as target. Data was obtained from three replicates. e Detection of SARS-CoV-2 in human swab samples. Ct-values of qPCR analysis (Orf1ab gene) of 81 samples are depicted on the X-axis with the true negative samples displayed as not determined (ND). See Supplementary Fig. S5 and Supplementary Table S2 for Ct-values of qPCR analysis of SARS-CoV-2 samples and respective Scope tool score. The data on the qPCR and SCOPE analysis was obtained from one replicate measurement. f One-pot LAMP-CRISPR limit of detection assay, using reconstituted Cmr-46, on a synthetic SARS-CoV-2 E-gene. Data was obtained from two replicates. All error bars in this figure represent the standard deviation of the mean. ‘Arb. units’ stands for arbitrary units. Source data are provided as a Source data file.

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