Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic

Figures

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  Fig. 1. Type III CRISPR-based RNA concentration enhances sequence-specific detection of RNA. a Schematic of type III CRISPR-based RNA concentration. RNase-dead type III CRISPR complex from Thermus thermophilus (e.g., TtCsmCsm3-D34A) is added to a sample to bind complementary “Target RNA”. The His-tagged complex is concentrated using nickel-derivatized magnetic beads and a magnet. b Sequence-specific RNA enrichment with TtCsmCsm3-D34A was tested using 25 nM 32P 5‘-end labeled RNA. Target or non-target RNA fragments were mixed with His-tagged TtCsmCsm3-D34A complex (125 nM) and concentrated with nickel-derivatized magnetic beads. Phenol-chloroform extracted RNAs from the supernatants and Csm-beads were resolved using UREA-PAGE. c Csm-based direct RNA detection using 3 μL of sample is compared to an assay where TtCsmCsm3-D34 is used to capture RNA at the same concentration from a 120 μL volume. Magnetic beads decorated with TtCsmCsm3-D34A are added to the sample. After concentrating beads with a magnet, the supernatant is decanted, and he pellet is resuspended in a reaction buffer with ATP to activate polymerase activity of Cas10. Polymerization products (e.g., cA3 and cA4) are used for the…

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  Fig. 2. Can1 and Can2 ancillary nucleases function as cA4-activated nucleases on plasmid DNA, ssRNA, and ssDNA. a Domain organization of Can1 and Can2 proteins. Can2 proteins have two domains – CARF and PD-(D/E)XK superfamily nuclease domain. Can1 is predicted to be derived from Can2 by gene duplication32. NLD – nuclease-like domain. b Maximum-likelihood phylogeny of 204 Can1 (CARF2 and PD-(D/E)XK nuclease domain) and 3121 Can2 proteins. The light gray numbers indicate bootstrap values. Previously studied effectors are marked with asterisks (*). Effectors chosen for purification and in vitro experiments are underlined. c–e Plasmid DNA (15 nM), ssRNA (425 nM), and ssDNA (15 nM) cleavage assay with TtCan1 (200 nM) in the presence of cA4 (50 nM). The reactions were incubated 15 min at 60 °C. n – nicked, lin – linear, sc – supercoiled plasmid. f–h Cleavage assays with AaCan2 (200 nM) in the presence of cA4 (50 nM). Assays were performed with 15 nM plasmid DNA, 425 nM ssRNA or 15 nM ssDNA for 15 min at 60 °C. n – nicked, lin – linear, sc – supercoiled plasmid. Source data are provided as a Source Data file.

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  Fig. 3. Incorporation of Can2 nuclease improves sensitivity of Csm-based RNA detection. a TtCsm6 (300 nM) and b AaCan2 (300 nM) cleavage assays with fluorescent ssRNA reporter (top) in the presence of varying cA4 activator concentrations (shown with colors). Data are shown as the mean (center line) of three replicates ± S.D. (ribbon). The optimal fluorescent reporter (top) was determined using RNA library screen in Supplementary Fig. 6. TtCsm RNA detection coupled with TtCsm6- (c) and AaCan2-based (d) assays were performed using samples with target RNA concentrations ranging from 107 to 104 copies/µL. Samples were prepared by spiking IVT fragments of SARS-CoV-2 N gene into total RNA extracted from nasopharyngeal swab patient sample negative for SARS-CoV-2. Cleavage of fluorescent RNA reporter was detected by measuring fluorescence every 10 s in a real-time PCR instrument (left). Data were plotted as mean of four replicates. Simple linear regression was used to calculate slopes for linear regions of the curves. Bars show mean values (n = 4) ± S.D. (right). Data was analyzed with one-way ANOVA followed by multiple comparisons to NTC sample using one-tailed post-hoc Dunnett’s test. ***p <…

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  Fig. 4. Incorporation of cA3-activated nucleases into Csm-based RNA detection assay. a The target bound TtCsm complex primarily generates cA4 and cA3. Schematic summarizes cA4- and cA3-dependent activities of nucleases biochemically tested. N/D – not detected; Asterisks (*) indicate nucleases that have sequences preferences (Supplementary Fig. 6). b CtNucC (15 nM) is activated by cA3 (20 nM) and cleaves plasmid DNA into short fragments in 15 min. c Deep sequencing of DNA fragments generated after 5 min of incubation with CtNucC revealed the preferential cleavage sites (ANNT). The reduced sequencing depth at the cut site is consistent with a cleavage mechanism producing 3′-overhangs that are removed by T4 DNA polymerase when sequencing library is prepared. d CtNucC (300 nM) cleavage assay with fluorescent dsDNA reporter across eight concentrations of cA3 (shown with colors). Data is shown as mean (center line) of four replicates ± S.D. (ribbon). e–g TtCsm RNA detection assays coupled with CtNucC (dsDNA reporter that include preferred cleavage sequence), AaCan2 (ssRNA reporter), and combination of AaCan2 and CtNucC (both reporters). Reactions were performed using samples with target RNA…

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  Fig. 5. TtCsm-based RNA capture directly detects SARS-CoV-2 in clinical samples. a Schematic of TtCsm-based RNA capture assay from patient sample coupled with AaCan2-based fluorescent detection. Two-step protocol includes incubation of TtCsm-beads with RNA sample at 60 °C followed by concentration of the magnetic beads, and addition of a buffer containing ATP, AaCan2 and a fluorescent RNA reporter (i.e., FAM-RNA-Iowa Black FQ). b RNA detection assay as shown in (a) was performed using samples with target RNA concentrations ranging from 107 to 102 copies/µL. Samples were prepared by spiking IVT fragments of SARS-CoV-2 N gene into in total human RNA (HEK 293T cells). Cleavage of fluorescent RNA reporter was detected by measuring fluorescence every 10 s in a real-time PCR instrument (left). Data were plotted as mean of three replicates ± S.D. (ribbon). c Simple linear regression was used to calculate slopes for linear regions of the curves. Bars show mean values (n = 3) ± S.D. (right). Data was analyzed with one-way ANOVA followed by multiple comparisons to NTC sample using one-tailed post-hoc Dunnett’s test. ***p < 0.001; **p < 0.01; *p < 0.05. d Patient samples (e.g., nasopharyngeal…