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

Figures

• 

  Fig. 1. In vitro RNase activity assays with the endogenous and reconstituted TtCmr complexes. a Schematic illustration of the different reconstituted TtCmr complexes used in the activity assays shown in panel c, pre-loaded with either the 46 (TtCmr-46), 40 (TtCmr-40) or 34 nt (TtCmr-34) crRNA (top strand). Red triangles indicate the anticipated cleavage sites (α, β, γ and δ) in the 4.5 target RNA (bottom strand, Supplementary Table S1) by the endoribonuclease activity of the Cas7 subunits. The target RNA was radiolabeled at the 5′ end with 32P γ-ATP (“P” in the red circle). b Denaturing PAGE analysis of the activity assay using a 5′ labeled target RNA complementary to the crRNA incubated with the endogenous TtCmr complex. A single stranded RNA marker (“M”) was used as size standards as indicated on the left. c Activity assays similar to panel b but using the reconstituted complexes. Discontinuous gel lanes are indicated by a dashed line. The results of the cleavage assays are representative results of three (b) or two (c) replicates (Supplementary Fig. 6). Source data are provided as a Source data file.

• 

  Fig. 2. Impact of complementarity in the 5′ handle and mismatches in the first spacer region on RNA targeting and cOA production. a Schematic illustration of the TtCmr complex bound to a target RNA (4.5 target RNA, Supplementary Table S1), showing the different subunits in different colors, crRNA (top strand) and target RNA (bottom strand). The target RNA was labeled at the 5′ end with 32P (“P” in the red circle). Red triangles indicate the cleavage sites within TtCmr. Highlighted in yellow are the first 5 nucleotides in the 5′ handle of the crRNA (nucleotides −1 to −5). b Different target RNAs (Supplementary Table S1) with matches to the 5′ handle were used in an activity assay and analyzed on denaturing PAGE. c Impact of 5′ handle complementarity on PPi release as a measure of COA production. d Similar schematic illustration as panel a with the first 7 nucleotides of the spacer region of the crRNA highlighted in yellow. e Similar activity assay as in panel b but with RNA targets (Supplementary Table S1) with single mismatches in the first 7 nucleotides of the spacer region of the crRNA. f Impact of mismatches in the target RNA with the first 7 nucleotides of the spacer region of the…

• 

  Fig. 3. A flexible seed region at the 3′ end of the crRNA. a Schematic illustration of the endogenous TtCmr complex. b Different target RNAs with segments mismatches were used in an activity assay and analyzed on denaturing PAGE. c Impact of target RNAs with mismatches in the indicated segments on the production of cOA. d Schematic overview of the 46 nt crRNA complex (TtCmr-46). e Similar to panel b, using the 46 nt crRNA complex (TtCmr-46). f Similar to panel c, using 46 nt crRNA complex (TtCmr-46). g Schematic overview of the 40 nt crRNA complex (TtCmr-40). h Similar to panel b, using the 40 nt crRNA complex (TtCmr-40). i Similar to panel c, using the 40 nt crRNA complex (TtCmr-40). Target RNA contain a 5’ end Cy5 label (red circle); mismatched segments are indicated with S1–S6. Red triangles indicate the cleavage sites within TtCmr. CAR segments are indicated in green, seed segments are indicated in red. The results of the cleavage assays and cOA production assays displayed in this figure are representative results of three independent experiments (Supplementary Fig. S6). Error bars represent the standard deviation of the mean. Source data are provided as a Source data file.

• 

  Fig. 4. Structural basis for flexible 3′ seed region in TtCmr complexes. a Overall structure of TtCmr complex with 46 nt crRNA (EMD-2898). b Overall structure of TtCmr complex with a 40 nt crRNA (EMD-2899). Complexes are colored as in Fig. 1. Boxed regions refer to close-up views shown in panels c and d. c Close-up view of the top of TtCmr46. d Close-up view of the top of TtCmr34. The ‘open’ seed region located at the 3′ end of the crRNA is more accessible than the upstream regions located towards the 5′ end, which are protected by Cmr5 subunits. The 3′ end of the crRNA is thus primed for propagating conformational changes and repositioning Cmr5 subunits along the complex upon target binding.

• 

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

• 

  Fig. 6. Schematic model of TtCmr target binding and subsequent activities. (1) TtCmr complex with bound crRNA is scanning for complimentary target RNA. (2) Target RNA binding is initiated at seed region, which induces a conformational change that allows further base pairing. (3) Full base pairing of target RNA to the crRNA, activating Cas10. (4) Target RNA is cleaved by Cas7 subunits and cOA is produced by Cas10. (5) Cleaved target RNA dissociates from TtCmr.