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

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.


Nucleic acid preparation a) Plasmids, ssDNA probe, and oligos
VirD2-SpCas9 and SpCas9-VirD2 clones were used to purify the fusion proteins. Mutant VirD2-SpdCas9 (dead SpCas9-VirD2), SpdCas9-VirD2 (VirD2-dead SpCas9), and SpdCas9-dVirD2 (dead SpCas9-dead VirD2) clones for expression of the respective fusion proteins were custom synthesized by GenScript. Guide RNAs for SpCas9 experiments were designed using SnapGene and ordered as gBlocks from Integrated DNA Technologies under the T7 promoter for in vitro transcription. The FAM-labeled ssDNA probe and biotin-labeled oligos were ordered from Integrated DNA Technologies. Sequences of the plasmids and oligos are listed in the supplementary file.

b) In vitro transcription of sgRNA
In vitro transcription was performed using TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific) following the manufacturer's instructions. Briefly, 10 L of 5X TranscriptAid Reaction Buffer, 20 L NTP mix, 10 L of the DNA template (annealed sgRNA gBlock and T7 promoter oligo), 0.5 L of RNase Out, 5 L of TranscriptAid Enzyme Mix, and 4.5 L of DEPC-treated water were incubated at 37°C for 8 hours. In vitro transcribed RNA was purified using Direct-zol RNA MiniPrep Kit (Zymo Research). Production of the proper size sgRNA fragments was confirmed on a 2% agarose gel run in Tris-Borate-EDTA buffer.

Protein purification
Protein purification was performed as previously described [13]. Briefly, a single colony of BL21(DE3) was grown in 2X-YT media and induced at 0.6 OD 600 with 0.3 mM IPTG and incubated at 18°C for 15 h at 180 rpm. Proteins were isolated using affinity purification column and further purified by size fractionation using the ÄKTA pure system (Cytiva).

Functional characterization of in-house produced enzymes a) SpCas9 nuclease activity assay of the fusion proteins
Target fragment (SARS-CoV-2, N-gene fragment) was amplified by PCR for SpCas9-based cleavage assays. Ribo-nucleoprotein particles of VirD2-SpCas9, SpCas9-VirD2, and catalytically dead mutants VirD2-SpdCas9, SpdCas9-VirD2 and SpdCas9-dVirD2 were assembled at 37°C for 10 minutes in 16.3 L reaction consisting of 250 nM of the respective protein, 250 nM sgRNA in the cleavage buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mM MgCl 2 ). Following the incubation, 3.7 L containing 150 ng of the target was added into the tube and the reaction was incubated for 1 hour at 37°C. The protein was denatured at 95C for 5 minutes, the reaction was cooled on ice for 3 minutes, and the DNA products were separated on a 2% agarose gel.

RT-RPA reactions with synthetic targets
RT-RPA was performed using the TwistAmp Basic kit following the manufacturer's instructions. Briefly, a well-mixed 47.5-L sample (1 l RNA template, 2.4 L of 10 M biotinlabeled forward and 2.4 L of unlabeled reverse primers, 29.5 L of Rehydration buffer, 0.5 L of SuperScript IV reverse transcriptase, 1 L of RNase H, 0.5 L of RNase Out, 10.2 µL H 2 0) was added to the lyophilized RPA reaction components (TwistAmp Basic) and homogenized by pipetting. Magnesium acetate (2.5 L of 280 mM) was added to each tube and mixed. The isothermal amplification was performed at 42C for 25 minutes. To confirm the DNA isothermal amplification, 10 L of the reactions were purified using QIAquick PCR Purification Kit and separated on a 1.5 % agarose gel.
Two reactions, 20 L each, were combined in a single tube to make 40 L preassembled reporter reaction.

VirD2-SpdCas9 detection assay and lateral flow assay
RT-RPA product (5 L) was mixed with 40 L of preassembled reporter. The 45-L reaction mix was incubated at 37C for 10 minutes followed by 1 minute at 60C. Following the incubation, 55 L of the running buffer (44.5 L of the HybriDetect Assay Buffer with 10.5 L of 10% BSA) was added directly into the reaction and mixed. Room temperature adjusted HybriDetect Dipsticks were placed into the tube containing the reaction mixture. Lateral flow strips were removed from the tube as soon as the control band appeared and the result was called within 10 minutes. Images of the strips were taken within 25 minutes after the beginning of the LFA.
The respective volume of RNA sample was added to the RT-RPA reactions. Nuclease-free water was used as the negative control. Following the RT-RPA reaction, 5 L of the product was transferred into the reaction containing the preassembled reporter complex and the samples were detected as previously described. The detection limit was considered as the concentration that could be successfully detected within 10 minutes of the LFA assay in all three replicates.

Validation of the developed protocol with SARS-CoV-2 clinical sample
RNA samples from SARS-CoV-2 RT-PCR positive (26 clinical samples) and negative (4 clinical samples) were used for evaluation of our Vigilant protocol. RNA (4 µL) was added to the RT-RPA reaction and 5 µL of the amplified product was used for detection in the next step. RT-qPCR was performed on Trizol based isolated RNA.

Supplementary Figure 1.
VirD2 alone is capable of cleaving ssDNA containing the specific RB recognition sequence. Following the cleavage of the oligonucleotide, VirD2 remains covalently bound to Tyr29 moiety, leaving three nucleotides of the original sequence. This property can be exploited to attach labels at the 3' end of oligonucleotide sequence bound to VirD2. Figure 2. A, B), Covalent binding of VirD2 and ssDNA probe. Biotin labeled probe harboring the T-DNA right border sequence was incubated with fusion proteins in the presence of Mg 2+ . VirD2-Cas9, Cas9-VirD2, VirD2-dCas9, dCas9-VirD2 bound to biotin labeled probe were detected by western blot. Biotin labeled probe, unlabeled probe, and dCas9-dVirD2 (no binding to RB sequence containing probe) were used as experimental control. Red arrow head indicated the immuno-detection of the biotin labeled probe bounded to fusion proteins. Selection of the optimal RT-RPA product volume for detection. Different volumes (0, 1, 2, 3, 4, 5 µL) of the RT-RPA product were added to the preassembled reporter complex. No template and no sgRNA were used as negative controls. D. Selection of the optimal duration for detection. RT-RPA product was incubated with preassembled reporter complex for different time periods. Incubation at 37 0 C for 10 minutes followed by 1 minute at 60 0 C results in robust, rapid and consistent appearance of the bands at the test line.   Log RNA Copy Number

Ct Value
Supplementary Figure 10. Copy number determination by RT-qPCR for Ct value relevance in clinical samples. Synthetic SARS-CoV-2 RNA template was used to determine the LOD by RT-qPCR using One-step RT-qPCR kit (Invitrogen). Ct value was determined using two independent sets primer sets, N1 and N2. Blank sample (no template) was used as negative control.  Figure 11. Reporter complex stability. Protein:sgRNA:FAM-probe were mixed in 1:1:1 ratio in 250, nM concentration and incubated at 37 0 C for 60 minutes. The reporter complex was stored at different temperatures for different periods of time. N-gene specific target and specific sgRNA were used as positive and no template samples was used as negative control.