Vigilant: An Engineered VirD2-Cas9 Complex for Lateral Flow Assay-Based Detection of SARS-CoV2

Abstract

Rapid, sensitive, and specific point-of-care testing for pathogens is crucial for disease control. Lateral flow assays (LFAs) have been employed for nucleic acid detection, but they have limited sensitivity and specificity. Here, we used a fusion of catalytically inactive SpCas9 endonuclease and VirD2 relaxase for sensitive, specific nucleic acid detection by LFA. In this assay, the target nucleic acid is amplified with biotinylated oligos. VirD2-dCas9 specifically binds the target sequence via dCas9 and covalently binds to a FAM-tagged oligonucleotide via VirD2. The biotin label and FAM tag are detected by a commercially available LFA. We coupled this system, named Vigilant (VirD2-dCas9 guided and LFA-coupled nucleic acid test), to reverse transcription-recombinase polymerase amplification to detect SARS-CoV2 in clinical samples. Vigilant exhibited a limit of detection of 2.5 copies/μL, comparable to CRISPR-based systems, and showed no cross-reactivity with SARS-CoV1 or MERS. Vigilant offers an easy-to-use, rapid, cost-effective, and robust detection platform for SARS-CoV2.

Keywords: COVID-19; CRISPR-Cas9; RT-RPA; SARS-CoV-2; VirD2; biosensors; dCas9; lateral flow assay; molecular diagnostics; nucleic acid detection; relaxases.

Figure 1

Vigilant platform for nucleic acid detection. (A) Schematic of reporter complex (FAM-probe–VirD2-dCas9–sgRNA) assembly. The ssDNA probe consists of a 25-bp T-DNA right border VirD2 recognition site at the 5′ end and a 5-bp dT stretch with FAM at its 3′ end. VirD2 recognizes the specific 25-bp motif in the ssDNA probe, cleaves it, and remains covalently bound to the 5′ end of the ssDNA probe. dCas9 uses the specific sgRNA to target the SARS-CoV-2 N gene. (B) Lateral flow assay. The streptavidin-coated line (T) captures biotin-labeled amplicons. In a sample containing SARS-CoV-2, the reporter complex bound to the target DNA will accumulate gold (Au) nanoparticle-labeled anti-FAM, resulting in the visual detection at the test line. The control line (C) is impregnated with anti-anti FAM antibodies that also accumulate Au nanoparticle-labeled anti-FAM and thus serve as a positive control. Detection of the positive samples is achieved by running the lateral flow after amplification and incubation with the reporter complex. The positive reaction is indicated by the presence of the lower (test) band while the upper band represents the control band. (C) Proof-of-concept of the Vigilant platform. SARS-CoV-2 N gene PCR amplicons were used as detection targets. PCR product (5 μL) was challenged with the preassembled reporter complex. ST (specific N-gene target), NST (nonspecific N-gene target), NSsgRNA (nonspecific sgRNA), NTC (no target control), (−) sgRNA (no sgRNA control), and (−) RC (no reporter complex). (D) Selection of the optimal fusion and probe. PCR product (5 μL) was challenged with the preassembled reporter complexes made with VirD2-Cas9 or Cas9-VirD2. Two probes, having 5 T nucleotides (probe 1) and 10 T nucleotides (probe 2) were used in the assembly of the reporter complex. Reactions with no sgRNA, no target control, or unlabeled target were used as controls. ST (specific N-gene target), NST (nonspecific N-gene target), NSsgRNA (nonspecific sgRNA), NTC (no target control). (E) Buffer composition optimization. Four buffers, having different compositions (see Materials and Methods in the Supporting Information) were used. Buffer 4 with BSA added to the running buffer was selected based on the enhanced signal detection and lower nonspecific background. ST (specific N-gene target), NST (nonspecific N-gene target), NSsgRNA (nonspecific sgRNA).

Figure 2

RT-RPA coupled with Vigilant for SARS-Cov2 detection. (A) SARS-CoV-2 RNA is reverse transcribed and amplified by RT-RPA using biotin-labeled primers. The biotin-labeled amplicon is then mixed with the preassembled reporter complex. Upon target recognition via the sgRNA, Cas9 remains stably bound to the DNA, yielding a complex that is labeled with both FAM (probe) and biotin (target). (B) RT-RPA product detection with the Vigilant platform. SARS-CoV-2 (synthetic) N-gene RT-RPA amplified product used as detection target. RT-RPA product (5 μL) was challenged with the preassembled reporter complex. Nonspecific N-gene target and nonspecific sgRNA, no target control, no sgRNA, and no reporter complex were used as controls. ST (specific N-gene target), NST (nonspecific N-gene target), NSsgRNA (nonspecific sgRNA), NTC (no target control), (−) sgRNA (no sgRNA control), and (−) RC (no reporter complex). (C) The Vigilant platform is compatible with VirD2-dCas9. Fusion proteins, VirD2-dCas9, dCas9-VirD2, and dCas9-dVirD2 were purified and evaluated for the Vigilant platform. RT-RPA product (5 μL) was challenged with the preassembled reporter complexes made with VirD2-dCas9 or dCas9-VirD2 or dCas9-dVirD2. VirD2-dCas9 demonstrated superior performance compared to the other two fusion proteins. ST (specific N-gene target), NST (nonspecific N-gene target), NSsgRNA (nonspecific sgRNA). (D) The Vigilant platform specifically detected SARS-CoV-2. RT-RPA was performed using SARS-CoV, SARS-CoV-2, MERS-CoV, TMV, and PVY as templates. RT-RPA product (5 μL) was challenged with the preassembled reporter complex. The Vigilant platform specifically detected only SARS-CoV-2.

Figure 3

Sensitivity and stability of the Vigilant platform for nucleic acid detection. (A) LoD determination with synthetic SARS-CoV2 RNA. A serial dilution (0, 1, 2.5, 7.5, 10, 50, and 100 copies per microliter) of SARS-CoV-2 synthetic RNA was prepared and amplified by RT-RPA using biotin-labeled primers. The biotin-labeled amplicon is then challenged with the preassembled reporter complex and visualized on LFA strips. (B) Stability of the preassembled Vigilant reporter complex. The reporter complex was stored at 4 °C, −20 °C, and room temperature for different time durations. SARS-CoV-2 (synthetic) N-gene RT-RPA amplified product (5 μL) was then used for detection. ST (specific N-gene target), NTC (no target control).

Figure 4

Validation of the Vigilant platform for SARS-CoV2 clinical samples. (A) Detection of SARS-CoV-2 in clinical samples. RT-RPA was performed for detection of SARS-CoV-2. SARS-CoV-2 RNA was isolated with the Trizol method. Samples with viral load (Ct value, 16–38) were detected with the Vigilant platform. The N-gene RT-RPA amplified product (5 μL) was subjected to the preassembled reporter complex. Samples with Ct value >38 were considered as negative. (B) Table representing experimental comparison of Vigilant, iSCAN, and RT-qPCR. (C) iSCAN method for the detection of SARS-CoV2 in clinical samples. An RT-LAMP-based iSCAN was performed for comparison with the Vigilant platform. (D) Schematic of POC utility of the Vigilant platform. After sample is collected from saliva or nasopharyngeal swab and transported in VTM, RNA is extracted and used as input for detection using Vigilant. In the first step RT-RPA with biotin-labeled primer is performed to amplify the viral genome. The resulting amplicons are then detected using the preassembled reported complex and visualized using LFA strips.