SpCas9 purification
BL21-CodonPlus (DE3)-RIL competent cells (Agilent Technologies 230245) were transformed with Cas9 plasmid (Addgene #67881) and inoculated in 25 mL of LB-ampicillin media. The bacteria culture was first allowed to grow overnight (37 °C, 220 rpm) and then transferred to 2 L of LB supplemented with ampicillin and 0.1% glucose until OD600 of ~0.5. Subsequently, the cells were induced with IPTG at a final concentration of 0.2 mM and maintained overnight at 18 °C. The bacteria cells were pelleted at 4500 × g, 4 °C for 15 min and resuspended in 40 mL of lysis buffer containing 20 mM Tris pH 8.0, 250 mM KCl, 20 mM imidazole, 10% glycerol, 1 mM TCEP, 1 mM PMSF, and cOmplete™ EDTA-free protease inhibitor tablet (Sigma-Aldrich 11836170001). This cell suspension was lysed using a microfluidizer and the supernatant containing Cas9 protein was clarified by spinning down cell debris at 16,000 × g, 4 °C for 40 min and filtering with 0.2 µM syringe filters (Thermo Scientific™ F25006). 4 mL Ni-NTA agarose bead slurry (Qiagen 30210) was pre-equilibrated with lysis buffer. The clarified supernatant was then loaded at 4 °C. The protein-bound Ni-NTA beads were washed with 40 mL wash buffer containing 20 mM Tris pH 8.0, 800 mM KCl, 20 mM imidazole, 10% glycerol, and 1 mM TCEP. Gradient elution was performed with buffer containing 20 mM HEPES pH 8.0, 500 mM KCl, 10% glycerol, and varying concentrations of imidazole (100, 150, 200, and 250 mM) at 7 mL collection volume per fraction. The eluted fractions were tested on an SDS–PAGE gel and imaged by Coomassie blue (Bio-Rad 1610400) staining. To remove any DNA contamination, 5 mL HiTrap Q HP (Cytiva 17115401) was charged with 1 M KCl and then equilibrated with elution buffer containing 250 mM imidazole. The purified protein solution was then passed over the Q column at 4 °C. The flow-through was collected and dialyzed in a 10 kDa SnakeSkin™ dialysis tubing (Thermo Fisher 68100) against 1 L of dialysis buffer (20 mM HEPES pH 7.5, 500 mM KCl, 20% glycerol) at 4 °C, overnight. Next day, the protein was dialyzed for an additional 3 h. in fresh 1 L of dialysis buffer. The final Cas9 protein was concentrated to 10 µg/µL using Amicon Ultra-15 Centrifugal Filter Unit, Ultracel-10 (Millipore Sigma UFC901008), aliquoted, and flash-frozen and stored at −80 °C.
eSpCas9 purification
The purification protocol was adapted from the manuscript associated with the eSpCas9 plasmid (Addgene #126769). Briefly, the eSpCas9 plasmid was transformed into BL21 Rosetta 2 (DE3) cells (Millipore EMD 71397) then grown in LB media with 10 µg/mL Kanamycin overnight at 37 °C. 10 mL of this culture was inoculated into 1 L of LB media with 10 µg/mL Kanamycin and grown to a final cell density of 0.6 OD600, then chilled at 18 . The protein was expressed at 18 °C for 16 h. following induction with 0.2 mM IPTG. The cells were centrifuged at 6000 × g for 15 min at 4 °C, resuspended in 30 mL of lysis buffer (40 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole, 1 mM TCEP) supplemented with cOmplete™ EDTA-free protease inhibitor tablet (1 tablet/30 mL; Sigma-Aldrich 11836170001), and then sonicated on ice. The lysate was cleared by centrifugation at 48,000 × g for 40 min at 4 °C, which was then bound to a 5 mL Mini Nuvia IMAC Ni-Charged column (Bio-Rad 7800812). The resin was washed extensively with a solution of 40 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole, and the bound protein was eluted by a solution of 40 mM Tris pH 8.0, 250 mM imidazole, 150 mM NaCl, 1 mM TCEP. 10% glycerol was added to the eluted sample and the His6-MBP fusion protein was cleaved by TEV protease (Addgene pRK793) (3 h at 25 °C). The volume of the protein solution was made up to 100 mL with buffer (20 mM HEPES pH 7.5, 100 mM KCl, 1 mM DTT). The cleaved protein was purified on a 5 mL HiTrap SP HP cation exchange column (GE Healthcare 17115201) and eluted with 1 M KCl, 20 mM HEPES pH 7.5, 1 mM DTT. The protein was further purified by size exclusion chromatography on a HiPrep 26/60 Sephacryl S-200-HR column (GE 17-1195-01) in 20 mM HEPES pH 7.5, 200 mM KCl, 1 mM DTT, and 10% glycerol. The eluted protein was confirmed by SDS–PAGE and SimplyBlue™ SafeStain (Invitrogen LC6060). The protein was stored at −20 °C.
Rep-X purification
E. coli Rep helicase was purified and crosslinked as previously described (Arslan et al.20). Briefly, the pET28(+) vector containing Rep-DM4 sequence was transformed into E. coli BL21(DE3) cells and grown in TB medium. When optical density reached OD600 = 0.6 the protein overexpression was induced with 0.5 mM IPTG. The cells were incubated overnight at 18 °C and harvested by centrifugation at 10,000 × g. Our Rep-DM4 contains 6x-His tag on its N-terminus for Ni-NTA affinity-column-based purification. The cell pellet was resuspended in a lysis buffer and cells were lysed with a sonicator. After binding Rep to Ni-NTA column and several washes, Rep was eluted with 150 mM imidazole-containing buffer. Rep concentration was kept below 4 mg/mL (~50 µM) to avoid aggregation.
Our Rep-DM4 mutant has four native cysteines removed (C18L, C43S, C167V, C612A), while C178 is kept and S400C mutation is introduced for crosslinking. C178 and C400 are linked with bismaleimidoethane crosslinker (BMOE) to lock Rep in a closed conformation and form Rep-X. Optimal crosslinking is achieved at Rep concentration between 20 and 25 µM, with Rep to BMOE ratio of 1:5. Excess imidazole and crosslinker were removed by overnight dialysis in the storage buffer (50% glycerol, 600 mM NaCl, 50 mM Tris, pH 7.6). The samples are stored at −80 °C. This method achieves nearly 95% crosslinking efficiency.
Generating synthetic ssRNA via in vitro transcription
For SARS-CoV-2 N-gene, perform PCR using 0.5 µL each of 10 µM forward and reverse primers (IVT_19n_N2_FWD, IVT_19n_N2_REV; primer sequences in Table S1). Mix with 4 µL of water, 0.5 µL of 2019-nCoV_N_Positive Control template plasmid (Integrated DNA technologies 10006625), and 10 µL of Q5 2× master mix (New England BioLabs M0494). Thermocycle at 98 °C for 30 s for initial denaturation, followed by 35 cycles of 98 °C for 10 s, 69 °C for 20 s, 72 °C for 30 s, then final extension of 72 °C for 2 min and 4 °C hold. Use QIAquick PCR purification kit (Qiagen 28104) to clean up PCR reaction and elute in 35 µl EB supplied with kit—agarose gel of PCR product should visualize band at 931 base pairs.
For in vitro transcription, use the HiScribe T7 kit (New England BioLabs E2050) by mixing 17 µL of purified PCR product with 3 µL of 10× reaction buffer, 8 µL of NTP mix, 2.5 µL of 50 mM DTT, and 2 µL of T7 RNA polymerase mix for a 30 µL total volume. Incubate at 37 °C at least 2 h. Then, add 20 µL of water and 1 µL of DNase I, mix, and incubate for another 37 °C for 15 min. Perform reaction cleanup using Monarch® RNA Cleanup Kit (New England BioLabs T2040). Measure RNA concentration using Nanodrop.
For bat-SL-CoVZC45 N-gene, perform the same protocol with IVT_bat_N2_FWD, IVT_bat_N2_REV primers (sequences in Table S1), with the SARS-CoV Control template plasmid (Integrated DNA technologies 10006624).
Making stock reagents (LAMP primer mix, cr/tracrRNA, buffers, and master mix) for CRISPR-based SENTINEL assay
Make 100 µL (RT)-LAMP primer mix by mixing 8 µL of FIP, 8 µL of BIP, 2 µL of F3, 2 µL of B3, 4 µL of LF, 4 µL of LB, and 56 µL of nuclease free water. All primers stocks are 100 µM in TE buffer, and sequences in Table S1.
Make stock 2× SENTINEL buffer by mixing 1-part 10× lambda exonuclease reaction buffer (New England BioLabs M0262), 1 part 100 mM NaCl, 1 part 100 mM MgCl2, and 2-part 10 mM ATP, resulting in a solution composed of 67 mM Glycine–KOH, 2.5 mM MgCl2, 50 µg/mL BSA, 2 mM ATP, 10 mM MgCl2, and 10 mM NaCl.
To make 10 µM cr/tracrRNA, anneal 3 µL of crRNA with 3 µL of tracrRNA (Integrated DNA Technologies), both at stock concentrations of 100 µM in Duplex buffer (Integrated DNA Technologies 11-01-03-01). Heat at 95 °C in a thermocycler with heated lid for 3 min, cool on benchtop for 5 min, then add 27 µL of Duplex buffer to make 30 µL total.
To make 10 µM Cas9, mix 5 µL of 10 µg/µL Cas9 with 25 µL of Dialysis Buffer (20 mM HEPES pH 7.5, 500 mM KCl, 20% glycerol; this is the storage buffer used from Cas9 purification) to make 30 µL total.
To make 10 µM Cpf1, mix 5 µl of 10 µg/µL Alt-R® A.s. Cas12a (Cpf1) Ultra (Integrated DNA Technologies 10001272) with 25 µL of dialysis buffer from Cas9 purification to make 30 µL total (Cpf1 and Cas9 are approximately the same molecular weight).
To make Cas9-based 1× SENTINEL master mix for one reaction, mix 80 µL nuclease free water with 100 µL 2× SENTINEL buffer. Then, add 2 µL of 10 µM annealed cr/trRNA and 1.6 µL of 10 µM Cas9, then mix again. After room temperature incubation for 30 min to form the Cas9–gRNA complex, add 10 µL of 1 µM Rep-X, 2 µL of Lambda Exonuclease (New England BioLabs M0262), then mix again. This is sufficient for 10 reactions. This master mix can be made in larger batches, aliquoted to smaller volumes, and stored in −20 or −80 °C.
To make Cpf1-based 1× SENTINEL master mix, replace Cas9 with 1.6 µL of 10 µM Cpf1. Rep-X is also not required and is omitted from the master mix.
For experiment with omission of Rep-X and/or Lambda Exonuclease, add equivalent volumes of water instead.
CRISPR-based SENTINEL assay on synthetic ssRNA or dsDNA
For a 20 µL reaction, mix 7 µL of nuclease free water, 2 µL of phosphorothiolated LAMP primer mix, and 10 µL of 2× LAMP master mix (New England BioLabs E1700) per reaction. Mix in 1 µL of diluted, synthetic ssRNA or dsDNA, then incubate at 65 °C for 30 min. The reaction can be scaled up to 50 µL. Afterwards, add in TE buffer for 1:20 dilution. Take 2 µL of this diluted (RT)-LAMP product, mix with 18 µl of 1× SENTINEL master mix, then leave at room temperature for 30 min. This is done twice—once with master mix containing on-target gRNA, and another with master mix containing non-target gRNA. Add in 180 µL of 1× PicoGreen solution (1 µL PicoGreen reagent with 200 µL TE buffer) (Thermo Fisher P7589), mix well, then load all to a well of Nunc™ F96 MicroWell™ Black Polystyrene Plate (Thermo Fisher 237105). Using excitation wavelength of 485 nm and emission wavelength of 528 nm, measure the sample fluorescence using a Synergy H1 plate reader (BioTek).
For the separate negative control, (RT)-LAMP of input without spiked-in ssRNA/dsDNA was used in SENTINEL with the non-target gRNA.
Restriction enzyme (AfeI) SENTINEL assay on synthetic ssRNA or dsDNA
Only the reaction master mix composition is modified from the CRISPR-based SENTINEL assay. Prepare reaction master mix by mixing 16 µL water, 2 µL CutSmart Buffer (New England BioLabs), 0.2 µL AfeI (New England BioLabs), and 0.2 µL Lambda Exonuclease (New England BioLabs). This can be used to react with 2 µL of diluted (RT)-LAMP product for the assay.
For the non-targeting condition, AfeI is replaced with equal volume of water.
For the negative control, (RT)-LAMP of input without spiked-in ssRNA/dsDNA was used in SENTINEL where AfeI is replaced with equal volume of water.
CRISPR-based SENTINEL assay on heat-inactivated viral particles
Gentamicin/Amphotericin B mixture was first made by mixing equal volumes of 50 mg/mL Gentamicin (Sigma-Aldrich G1397) with 250 µg/mL Amphotericin B (Sigma-Aldrich A2942), then filter-sterilized using a 0.22 µm pore size filter unit. Viral transport media (VTM) was prepared by mixing 500 mL of Hanks balanced salt solution (HBSS), 10 mL heat-inactivated FBS (Corning), and 2 mL of filter-sterilized Gentamicin/Amphotericin B mixture.
Supernatant from SARS-CoV-2-infected Vero cell culture with 10E7 viral particles per µL was heated at 65 °C for 1 h for virus inactivation. Serial dilutions were performed using VTM. 1 µL of the dilution was mixed with 4 µL pooled human saliva (Innovative Research IRHUSL5ML) to simulate virus presence in human saliva. This was mixed 1:1 with QuickExtract™ DNA Extraction Solution (Lucigen QE09050), then immediately heated to 95 °C for 5 min. 1 µL of this final solution was used in a 20 µL SENTINEL reaction, with the remainder of the protocol identical to the section “CRISPR-based SENTINEL assay on synthetic ssRNA or dsDNA”.
Patient consent statement and study cohort
From April 21, 2020 to July 16, 2020, non-hospitalized adults who were self-isolating after receiving a positive NP SARS-CoV-2 real-time reverse-transcription polymerase chain reaction (rRT-PCR) result from the Johns Hopkins Medical Microbiology laboratory were approached for participation by telephone using a verbal consent script. Obtaining signed informed consent form for subjects enrolled in this study was not initially feasible for study staff due to the contagious nature of COVID-19 being studied under this protocol. Instead, the study staff used a consent waver and obtained verbal consent. Inclusion criteria were age ≥18 years, able to receive study materials while remaining in isolation, and able and willing to perform self-collection of specimens. This protocol and verbal consent were approved by the Johns Hopkins University School of Medicine Institutional Review Board (IRB), and are identical to that of Manabe et al. (2020)32 and Antar et al. (2021)33. All procedures were in accordance with the ethical standards of the Helsinki Declaration of the World Medical Association.
Specimen collection
Participants were mailed a sample collection kit that included an international air transport association (IATA)-approved biologic sample container as well as sample collection materials and written instructions for sample collection. Participants self-collected mid-turbinate nasal and oropharyngeal (nasal-OP) swabs; both swabs were placed in 3 mL viral transport medium. All samples were immediately placed in the IATA container and stored in the participant’s freezer before shipping. Participants self-collected samples on the day they received the collection materials (day 0) and then subsequently on study days 3, 7, 14. On day 14, the participant shipped the collected samples on ice-cold packs to Johns Hopkins University for analysis using an overnight courier service. The procedure was identical to that of Manabe et al. (2020)32 and Antar et al. (2021)33.
Blinding schema
Individual 1 has information on ground-truth quantitative PCR readout for each patient sample. 25 SARS-CoV-2 positive samples and 25 SARS-CoV-2 negative samples were randomly placed in a freezer box by Individual 1. When Individual 1 was not present, Individual 2 removed the samples from the freezer box and performed the SENTINEL assay. Individual 1 sends the SENTINEL results to Individual 2 for verification.
SENTINEL assay on patient nasopharyngeal swabs
35 µL of patient nasopharyngeal swabs was mixed 1:1 with QuickExtract™ DNA extraction solution (Lucigen QE09050), then immediately heated to 95 °C for 8 min. 2 µL of this final solution was used in a 50 µL SENTINEL reaction. 45 min was used for the RT-LAMP step, and detection was performed on the SpectraMax i3x plate reader (Molecular Devices). Everything else was identical to the section “CRISPR-based SENTINEL assay on synthetic ssRNA or dsDNA”.
Computation of SENTINEL score
Reaction A corresponds to the on-target SENTINEL reaction (on-target Cas/gRNA or on-target restriction enzyme). Reaction B corresponds to the non-target SENTINEL reaction (non-target Cas9/gRNA or water in the place of restriction enzyme). Reaction C corresponds to the negative control—the sample expected to have no target nucleic acid, exposed to the non-target SENTINEL reaction. A–C will be the results of Reactions A–C, respectively, as fluorescence measurements (in arbitrary units) on a plate reader.
The first part of the SENTINEL score is fractional reduction in fluorescence with on-target versus non-target Cas9/gRNA, i.e., 1–A/B. Next, this value is scaled by the fractional increase in DNA quantity due to LAMP amplification (B/C). Together, the SENTINEL score is computed using the following formula: (1−A/B) * (B/C).
Statistics and reproducibility
All statistical analyses were conducted on either Microsoft Excel or GraphPad Prism 9. All data for statistical analysis were numerical, and a sample size and/or replicates of at least 3 were used for hypothesis testing using Student’s t-test. Replicates were all ‘biological’, in that that each sample of the replicate was independently conducted and in separate test tubes.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
