key: cord-1006787-fe8cjf2e
authors: Santiago-Frangos, Andrew; Hall, Laina N.; Nemudraia, Anna; Nemudryi, Artem; Krishna, Pushya; Wiegand, Tanner; Wilkinson, Royce A.; Snyder, Deann T.; Hedges, Jodi F.; Cicha, Calvin; Lee, Helen H.; Graham, Ava; Jutila, Mark A.; Taylor, Matthew P.; Wiedenheft, Blake
title: Intrinsic Signal Amplification by Type-III CRISPR-Cas Systems Provides a Sequence-Specific SARS-CoV-2 Diagnostic
date: 2021-05-27
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2021.100319
sha: 372934d65163b6014fedf9014f6c8c3c2ba894e5
doc_id: 1006787
cord_uid: fe8cjf2e

There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here we repurposed the type III CRISPR-Cas system for sensitive and sequence specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design 10 guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/μL for a single guide RNA, to 106 copies/μL for 10 guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/μl and is completed using patient samples in less than 30 minutes.

Introduction and the wildtype Csm complex could detect the SARS-CoV-2 RNA at concentrations above 10 8 copies per reaction, and neither complex cross-reacted with the SARS-CoV-1 RNA at the highest concentrations tested. The RNase-dead TtCsm complex was roughly 3-fold more sensitive than wildtype, with an LoD of ~10 7 copies per reaction.

In addition to fluorometric detection, we also developed a colorimetric RNA detection method that utilizes a pH change that occurs during nucleotide polymerization (Figures 1D and S3) . Specific recognition of SARS-CoV-2 by RNase-dead TtCsm complex, activates Cas10. Cas10 polymerizes ATP [25] [26] [27] [28] , releasing one proton per incorporated nucleotide. Cas10-generated protons acidify the solution and change the color of a pH indicator (i.e. Phenol Red) from fuchsia through orange (10 10 RNA copies), to yellow (10 11 RNA copies). Similarly, we developed a visible fluorometric detection method that relies on the sequestration of metallic ions by pyrophosphate. The metal indicator Calcein is initially quenched by bound Mn 2+ ions 33 . In addition to the cyclic oligoadenylates and protons, Cas10 polymerase generates one pyrophosphate per ATP polymerized. Pyrophosphate forms an insoluble precipitate with Mn 2+ , which unquenches Calcein. Free Calcein is then bound by excess Mg 2+ , forming a fluorescent complex that can be seen by eye or with a UV lamp in less than 10 minutes (Figures 1E and S4 ).

The LoD using crRNA N1 is between 10 7 and 10 8 copies of IVT RNA per µL, which is insufficient to be clinically relevant (Figures 1C and 2C) . To identify other guides that might outperform or complement the activity of crRNA N1 we aligned 45,641 SARS-CoV-2 genomes available from GISAID 34 . These alignments were used to select guides based on four key criteria. First, each target sequence had to be more than 99% identical among the available SARS-CoV-2 genomes. Second, complementarity between the target and the crRNA was not allowed to extend beyond the spacer sequence (guide), and into repeat derived portions of the crRNA that have been shown to suppress Cas10 activity 27 .

Third, we targeted regions of SARS-CoV-2 that were different by at least two-nucleotides in SARS-CoV-1 and MERS-CoV. Fourth, the list of target sequences was pruned to remove guides with similarity to human mRNA sequences, or common oral and respiratory pathogen sequences (E-value < 1000). Finally, we focused on target sequences located 3' of the ORF3a gene, which are present on both the viral genome and on subgenomic RNAs generated during infection. In total, we designed crRNAs targeting 10 different locations on the SARS-CoV-2 genome (Figure 2A and S1; Table S1 ).

To determine how each of these guides perform, we measured sequence specific detection of RNA using a fluorometric reporter assay (i.e., FAM-RNA-Iowa Black FQ) ( Figure 2B) . Most of the crRNAs provide similar sensitivity, however crRNA N1 and crRNA N9 generated significantly more signal than the next best complex tested (p-value<0.0001). We then tested crRNA N1 and crRNA N9 on RNA isolated from the nasal swab of an infected patient ( Figure 2C ). Both crRNA N1 and crRNA N9 guided complexes generate a similar signal for either IVT RNA or RNA isolated from a SARS-CoV-2 positive patient (i.e., 2-to 3-fold increase in signal by 5 minutes, relative to first timepoint). The LoD for crRNA N1 and crRNA N9 is 10 7 copies/µL ( Figure 2D ) (p-value<0.0001).

Fozouni et al., recently showed that multiplexing Cas13 (i.e., combining multiple guides into a single reaction) improves the sensitivity of SARS CoV-2 detection 19 . We reasoned that similar benefits might be possible for Csm-based detection. To test this idea, we combined 10 of the guides (2.5 nM each) into a single multiplexed reaction. Multiplexing 10 guides improves the sensitivity of TtCsm-mediated detection of SARS-CoV-2 RNA isolated form the nasal swab of a positive patient by approximately 10 times (Figures 2C and D) . However, the sensitivity of direct detection appears to increase additively with the number of TtCsm complexes, which precludes direct detection of RNA at concentration that are clinically relevant.

Csm-based detection is currently not sensitive enough to directly detect SARS-CoV-2 in all patients capable of spreading the infection, which requires an LoD of 10 3 RNA copies/µL 1, 2, 35, 36 (Figure 2C ).

To decrease the LoD of a type III CRISPR-based diagnostic to 10 3 RNA copies/µL or lower, we incorporated an upstream nucleic acid amplification technique ( Figure 3A) . First, SARS-CoV-2 genomic RNA is reverse transcribed (RT) into DNA, which is then amplified by LAMP using primers that flank regions of the SARS-CoV-2 genome targeted by crRNA N1 and crRNA N9 . One of the LAMP primers incorporates a T7 promoter into the amplified DNA, which is then used for in vitro transcription (T7) and detected by TtCsm (Figures 3A and S5 ; Table S4 ).

To confirm the specificity of TtCsm-based detection, we tested SARS-CoV-2 alongside a panel of eight other oral and respiratory pathogens, including coronaviruses SARS-CoV-1, Middle East respiratory syndrome coronavirus (MERS-CoV), Human coronavirus HKU1 and Human coronavirus NL63 ( Figure 3B ). These samples resulted in background signal similar to the no template control (NTC) ( Figure 3B ). In contrast, SARS-CoV-2 RNA results in a 4-5-fold increase in signal.

To determine the LoD of RT-LAMP-T7-Csm, we tested 20 replicates of 2-fold serial dilutions ranging from ~100-400 copies/µL SARS-CoV-2 RNA ( Figure 3C ). The LoD of RT-LAMP-T7-Csm is 198 copies/µL SARS-CoV-2 RNA (20/20 replicates), in an assay that relies on a 29-minute RT-LAMP step, followed by a 1-minute T7-Csm fluorometric detection reaction ( Figure 3D ).

To further validate this method, we next tested RNA extracted from 56 nasopharyngeal swab samples taken from patients that had previously been tested using RT-qPCR. Of the 56 samples tested, 46 were positive for SARS-CoV-2 and 10 were negative by RT-qPCR ( Figure 3E ). Using two different crRNA guides, we demonstrate that the type III CRISPR system has a specificity (negative predictive agreement) of 100%, as well as a positive predictive agreement of 100% for nasopharyngeal swab samples with 100-200 copies/µL SARS-CoV-2 RNA as determined by RT-qPCR (Figures 3E and  S5) . Whole genome sequencing revealed three of the patient samples used here belonging to the B.1.1.7. lineage. These genome sequences have been deposited in GISAID (Accession IDs: EPI_ISL_1081321, EPI-ISL_1081322, EPI_ISL_1081323) 34 . Importantly, the B.1.1.7. variants were J o u r n a l P r e -p r o o f positively identified by RT-LAMP-T7-Csm with both N1 and N9 crRNA guides ( Figure 3E ; N1, red circles; N9, blue triangles).

Collectively, this work demonstrates that sequence specific detection of viral RNA by the type III CRISPR-Cas complex triggers the synthesis of cA 4 , pyrophosphate, and protons, each of which are detectable within 1 to 30 minutes, using colorimetric or fluorometric methods (Figure 1) . Coupling RT-LAMP to T7 transcription and Csm-based detection creates a rapid (<30 minutes) testing protocol with attomolar sensitivity and high specificity. Moreover, we show that this protocol is capable of detecting the B.1.1.7 SARS-CoV-2 variant and we anticipate that the guide design criteria described here could be applied for specific detection of other pathogens. Further, the RT-LAMP and T7-Csm reactions occur in similar buffers and at similar temperatures, therefore it is possible that additional modifications that either increase the efficiency of RT-LAMP at 55°C (i.e. re-designing primer sets), or that increase the efficiency of T7-Csm at 65°C (i.e. a more thermostable RNA polymerase), could enable an integrated one-pot diagnostic.

While the LoD for RT-LAMP-T7-Csm is 1-1.5 orders of magnitude higher than FDA EUA approved DETECTR and SHERLOCK assays (20 and 6.75 copies/µL respectively), the time to result is ~30% faster, which would translate to a higher throughput of samples analyzed per unit of machine runtime. Sensitivity was strategically sacrificed for time in this proof-of-concept application for optimal detection of infectious SARS-CoV-2 patients, which requires an LoD of 1000 copies/µL 1,2,35,36 .

However, an increase in the length of the RT-LAMP step (i.e. to 40 minutes as is used in SHERLOCK), would likely increase the sensitivity. Additional modifications that could further increase sensitivity include, optimization of RT-LAMP primer sets, a screen for more active type III Csm or Cmr surveillance complexes, or a screen for CARF-fusion effectors that do not cleave their cyclic oligoadenylate ligands 37 . Alternatively, more sensitive methods of measuring Cas10-generated protons, such as ion-sensitive field-effector transistors used in label-free sequencing by synthesis technologies, could decrease the LoD.

Type V (Cas12-based) and type VI (Cas13-based) CRISPR-based diagnostics hinge completely on their collateral nuclease activity [13] [14] [15] [16] [17] 38 . The Cas10 subunits of Csm complexes possess an analogous nuclease activity triggered upon target binding 21, 29, [39] [40] [41] . However, here we focused on the NTP polymerase activity of activated Csm complex, which enables readouts based on several different chemistries (Figure 1) . Further, there are a rich resource of naturally occurring downstream effector proteins that have evolved to be activated by the Cas10 polymerized NTP products, that possess a wide range of enzymatic activities. These effectors include RNases, DNases, putative proteases, nitrilases, adenosine deaminases and adenylate cyclases 42 . Future efforts are aimed at incorporating complementary effectors with the goal of reducing the LoD for direct detection of RNA in clinical samples.

This study demonstrates a proof-of-concept for a sensitive, specific and rapid diagnostic based on the type III CRISPR system. The current implementation requires pre-amplification using RT-LAMP, followed by T7-transcription and type III detection (T3D). Clinical implementation of this approach will benefit from changes that eliminate the need for RT-LAMP and T7-amplification, improvements that limit sample handling, as well as a better understanding of the base pairing requirements that activate the Cas10 polymerase 16, 19, 26, 30, 32 . Improvements will benefit from screening additional guides, testing other type III complexes for accelerated polymerase activity, and incorporating other ancillary nucleases or other effectors in a way that boosts the sensitivity and reduces the time to result. 

One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in science. One or more of the authors of this paper self-identifies as a member of the LGBTQ+ community. One or more of the authors of this paper received support from a program designed to increase minority representation in science. While citing references scientifically relevant for this work, we also actively worked to promote gender balance in our reference list. and SARS-CoV-1 N-gene (black squares). Cyclic tetra-adenylate (cA 4 ) activates TtCsm6, which is a nonsequence specific ancillary nuclease ( Figure S2) . Activated TtCsm6 cleaves an RNA tether, which links a fluorophore (star) to a quencher (gray hexagon). TtCsm Csm3-D34A N1 (right graph) exhibits a LoD 3-fold lower than wildtype TtCsm N1 (left graph) and retains specificity for SARS-CoV-2 RNA. The mean of three technical replicates are shown, error bars represent ± 1 standard deviation. (D) Colorimetric detection of SARS-CoV-2 RNA by TtCsm Csm3-D34A N1 complex using a pH sensitive dye (i.e., Phenol Red). Reactions were incubated for 30 minutes at 60°C. Technical replicates are shown in Figure S3 . (E) Visible fluorometric detection of SARS-CoV-2 RNA by TtCsm Csm3-D34A N1 complex using Calcein. Reactions were incubated for up to an hour at 60°C. Technical replicates and kinetics are shown in Figure S4 . Table S1 and S3). Mean and standard deviation of two technical replicates is shown. (C) Direct detection of SARS-CoV-2 genomes in RNA extracted from patient samples by 25 nM TtCsm Csm3-D34A N1 or N9, or a mixture of ten different TtCsm Csm3-D34A complexes each at 2.5 nM, via a reporter RNA-based assay. RNA extracted from a patient with a high viral load (5x10 8 copies/µL as determined by RT-qPCR) was diluted into RNA extracted from patients lacking a SARS-CoV-2 infection. Mean and standard deviation of three technical replicates is shown. (D) Slopes of increasing fluorescence, measured in panel 2C, were calculated by simple linear regression. The calculated slope and ± 95% confidence intervals are shown. Positive RNA slopes were compared to the negative swab RNA slope by an F-test: ****p < 0.0001, ns = not significantly higher than negative swab RNA control. 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the leader contact, Blake Wiedenheft (bwiedenheft@gmail.com).

Plasmids generated in this study are available upon request.

Three sequenced SARS-CoV-2 genomes have been uploaded to GISAID (IDs EPI_ISL_1081321, EPI_ISL_1081322, EPI_ISL_1081323) and NCBI (MW940884, MW940885, MW940886). Genome IDs are listed in the Key Resources Table. The script used to design TtCsm guide RNAs specific to SARS-CoV-2 genomes is available at https://github.com/WiedenheftLab/Type-III_crRNA_Design. 

Bacterial strains Escherichia coli DH5α (Thermo Fisher Scientific) cells were used to amplify plasmids used in this paper. E. coli BL21 DE3 (NEB) cells were used to express proteins used in this paper. E. coli were grown in LB (Lennox) media at either 37°C, or 16°C after induction of protein expression with 0.5 mM IPTG (isopropyl-β-D-thiogalactoside).

Previously published LAMP primers (Eurofins) were designed to amplify the SARS-CoV-2 N-gene 13 . Target SARS-CoV-2 and SARS-CoV-1 RNAs were in vitro transcribed with MEGAscript T7 (Thermo Fisher Scientific) from PCR products generated from pairs of synthesized overlapping DNA oligos or using SARS-CoV-2 genome as a template ( bacterial and fungal pathogens were used as is, or resuspended in 1x TE (10 mM Tris-HCl pH 7.5, 1 mM Ethylenediaminetetraacetic acid (EDTA)) to ~1x10 6 genomes/µL (Table S5) .

Expression vectors for Thermus thermophilus type III-A csm1-csm5 genes, pCDF-5xT7-TtCsm were purchased from Addgene (plasmid # 128572) 45 . pCDF-5xT7-TtCsm was used as a template for sitedirected mutagenesis to mutate the Csm3 residue D33 to alanine (D33A) to inactivate Csm3mediated cleavage of target RNA (pCDF-5xT7-TtCsm Csm3-D34A ) 29 . The CRISPR array in pACYC-TtCas6-4xcrRNA4.5 (Addgene plasmid # 127764) 45 was replaced with a synthetic CRISPR array (GeneArt) containing five repeats and four identical spacers, designed to target the N-gene of SARS-CoV2 (i.e., pACYC-TtCas6-4xgCoV2N1). TtCas6 was PCR amplified from the pACYC-TtCas6-4xcrRNA4.5 plasmid and cloned between the NcoI and XhoI sites of pRSF-1b (MilliporeSigma) (pRSF-TtCas6). The CARF-HEPN nuclease TtCsm6 was expressed from pC0075 TtCsm6 His6-TwinStrep-SUMO-BsaI (Addgene plasmid # 115270) 14 .

Expression and purification of the TtCsm complex was performed as previously described with minor modifications 45 . Briefly, the crRNA plasmid (e.g. pACYC-TtCas6-4xgCoV2N1) was co-transformed with pRSF-TtCas6 and either pCDF-5xT7-TtCsm or pCDF-5xT7-TtCsm Csm3-D34A into Escherichia coli To screen guide RNAs in a high throughput format, ten TtCsm complexes were first crudely purified. 8 mL cultures of E. coli BL21-DE3 cells transformed with pTtCsm and pT7-5xCRISPR-Cas6 were grown at 37°C and 250 RPM in LB media with selective antibiotics until they reached an OD 600 reading of 0.4. Protein expression was then induced with the addition of 0.5 mM IPTG to the media, and cells were grown overnight at 16°C. Cells were collected by centrifugation at 4000 RPM, and cell pellets were resuspended in 250 µL of Ni-NTA Equilibration buffer (PBS; 100mM sodium phosphate, 600mM sodium chloride), 0.05% Tween™-20 Detergent, 30mM imidazole; pH 8.0). Resuspended cells were sonicated twice for twenty seconds, then clarified by centrifugation at 15,000 rpm for 20 minutes at -4°C to remove cellular debris. The lysate was then heat-treated at 55°C for 45 minutes, and re-clarified by centrifugation at 15,000 rpm, for 30 mins at 4°C. TtCsm was then purified using HisPur Ni-NTA magnetic beads (Thermo Fisher Scientific) according to the manufacturers recommendations, but with modified wash (25 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween-20, 1 mM TCEP) and equilibration (25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP) and elution buffers (25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, 300 mM Imidazole). TtCsm complex concentration was quantified on a Nanodrop (Thermo Fisher Scientific).

Fluorescent CRISPR-Csm based detection For experiments shown in Figure 1C , RNA was extracted from nasopharyngeal swabs derived from patients that tested negative for SARS-CoV LoD standards were prepared by diluting SARS-CoV-2 RNA into RNA extracted from COVID-19negative patient nasopharyngeal swabs. Concentrations were determined with RT-qPCR using a standard curve generated from 10-fold dilution series (1x10 6 -1x10 0 ) of IVT fragment.

Sequencing of SARS-CoV-2 RNA isolated from patient samples SARS-CoV-2 genomic RNA isolated from patient samples was sequenced as previously described 47 .

In brief, 10 µL of SARS-CoV-2 genomic RNA extracted from nasopharyngeal patient swabs was first reverse transcribed with SuperScript IV (ThermoFisher) according to the manufacturer's instructions. The ARTIC Network protocol was followed to generate a sequence amplicon library covering the whole SARS-CoV-2 genome on Oxford Nanopore using a ligation sequencing kit (Oxford Nanopore, SQK-LSK109) (https://artic.network/ncov-2019) 48, 49 . Two multiplex PCR reactions were performed with primer pools described in the ARTIC nCoV-2019 V3 Panel (Table S6) , amplified with Q5 DNA Polymerase (NEB). The two resulting amplicon pools for each patient sample were then combined and used for library preparation. Samples were end repaired (NEB, E7546) and then barcoded using Native Barcoding Expansion Kits (Oxford Nanopore, EXP-NBD104 and EXP-NBD114). Barcoded samples were pooled together and then Nanopore adaptors were ligated.

The multiplexed library was loaded onto the MinION flowcell, and a total of 0.3 Gb of raw sequencing data was collected per patient sample. Raw Nanopore reads were base-called in high-accuracy mode (Oxford Nanopore, MinKNOW), and further analyzed using the ARTIC bioinformatic pipeline for COVID-19 (https://artic.network/ncov-2019) 50 . Consensus sequences were uploaded to GISAID (https://www.gisaid.org/), IDs: EPI_ISL_1081321, EPI-ISL_1081322, EPI_ISL_1081323 34 . These three SARS-CoV-2 genome sequences were identified as members of the B.1.1.7 lineage by an automated lineage assigner 51 (https://github.com/hCoV-2019/pangolin).

All experiments were performed in triplicate or duplicate and error is reported as ± 1 standard deviation. The merged datasets of replicates of fluorescence kinetics of direct Csm-based detection of SARS-CoV-2 RNA in patient samples was fit to a simple linear regression, in Prism 9 (Graphpad).

The fitted slopes of SARS-CoV-2 RNA-containing patient samples were compared pairwise to the negative swab RNA control by an F-test, ****p < 0.0001. Figure S1 . Purification of TtCsm complexes and TtCsm6 nuclease. Related to Figures 1-3. (A) Size exclusion chromatography (SEC) profiles of TtCsm WT and TtCsm Csm3-D34A complexes loaded with different crRNA guides. SEC performed using a Superose 6 Increase 10/300 GL size-exclusion column (Cytiva). Normalized absorbance (mAU) was measured at 260nm (red) and 280nm (blue). Fractions 9 up to16 of each SEC purification were collected, concentrated, and stored at -80°C. (B) The fractions were combined, concentrated and run on an SDS-PAGE. All five Csm proteins are present and the intensities of each band correspond with our understanding of the protein stoichiometry of the assembled TtCsm complex. (C) RNA was isolated from the pooled and concentrated SEC fractions. Denaturing urea polyacrylamide gel of nucleic acids associated with each TtCsm complex. The full-length crRNA intermediate is expected to be 76 nucleotides (nts) long. (D) SEC profile of TtCsm Csm3-D34A N1 complex. Six successive fractions, representing the entire peak, were collected concentrated and stored separately. (E) Sequence-specific activation of Cas10 was estimated for each of the six fractions in panel D. 32 P-ATP polymerization was measured using thin-layer chromatography (TLC). 500 nM TtCsm Csm3-D34A N1 complex was incubated with 10 10 copies of target RNA, 50 µM ATP and 10 nM α 32 P-ATP, at 60°C for 1 hour. Nucleic acids were phenol-chloroform extracted from each reaction and spotted on a silica gel TLC plate coated with fluorescent indicator F254, developed in solvent (0.2 M ammonium bicarbonate pH 9.3, 70% ethanol, 30% water). An unlabeled cA4 standard (Axxora) was run on the same TLC plate, in a parallel lane, and was visualized by illumination with a handheld shortwave (254 nm) UV lamp (Analytik Jena) and a Galaxy S9 phone (Samsung). One of the two major 32 P-labelled products generated by target RNA-bound TtCsm complex migrates similarly to the cA4 standard. All TtCsm complexes polymerize similar amounts of α 32 

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Highlights • Sequence-specific recognition of RNA by CRISPR Csm complex activates Cas10 • Cas10 polymerizes ATP to make cyclic oligonucleotides, pyrophosphates and protons • Cas10's rapidly amplified products are detectable in 1-30 minutes • RT-LAMP can be coupled to T7-Csm to rapidly and sensitively detect SARS-CoV-2 RNA eTOC Recognition of a complementary target RNA by the type III CRISPR systems uniquely triggers the activation of a CRISPR-associated polymerase domain in Cas10. The polymerase generates oligo Adenylates, protons and pyrophosphates. Santiago-Frangos et al. repurposed the type III CRISPR-Cas system for sensitive and sequence specific detection of SARS-CoV-2 by developing

We are grateful to members of Bozeman Health that provided deidentified patient samples. 

J o u r n a l P r e -p r o o f