Differential Detection of the Tobamoviruses Tomato Mosaic Virus (ToMV) and Tomato Brown Rugose Fruit Virus (ToBRFV) Using CRISPR-Cas12a

CRISPR/Cas12a-based detection is a novel approach for the efficient, sequence-specific identification of viruses. Here we adopt the use of CRISPR/Cas12a to identify the tomato brown rugose fruit virus (ToBRFV), a new and emerging tobamovirus which is causing substantial damage to the global tomato industry. Specific CRISPR RNAs (crRNAs) were designed to detect either ToBRFV or the closely related tomato mosaic virus (ToMV). This technology enabled the differential detection of ToBRFV and ToMV. Sensitivity assays revealed that viruses can be detected from 15–30 ng of RT-PCR product, and that specific detection could be achieved from a mix of ToMV and ToBRFV. In addition, we show that this method can enable the identification of ToBRFV in samples collected from commercial greenhouses. These results demonstrate a new method for species-specific detection of tobamoviruses. A future combination of this approach with isothermal amplification could provide a platform for efficient and user-friendly ways to distinguish between closely related strains and resistance-breaking pathogens.


Plant Materials and Virus Inoculation
Tomato (Solanum lycopersicum L. cv. Moneymaker) (LA2706) were grown in soil in a light-and temperature-controlled chamber at 25 °C with a 16 h light/8 h dark regime. Three-week-old tomato plants were used for mechanical inoculation of ToMV and To-BRFV. ToMV was derived from the infectious clone pTLW3 [13]. For ToBRFV infection we used the Israeli isolate of ToBRFV, ToBRFV-IL (KX619418.1). Plants were dusted with carborundum powder prior to inoculation, and were then rubbed with a phosphate buffer solution (0.01 M, pH 7.0) supplemented with crushed leaves from ToMV-or ToBRFVinfected tomato plants. After three weeks, samples were collected from young leaves showing viral symptoms, distant from the site of inoculation.
For field sample analysis, tomato leaves with mosaic symptoms were collected from two commercial greenhouses in Azriel village, Israel, in December 2020. , RNA is extracted from the infected leaf. (C) Amplification of part of the viral genome using genus-specific RT-PCR primers. (D) Using species-specific crRNA, Cas12a-crRNAs specifically target the sequence of ToBRFV or ToMV. (E) Activation of Cas12a by sequencespecific binding, which triggers the degradation of the ssDNA probe and releases the fluorophore (F) from the quencher (Q) to emit the fluorescent signal.

Plant Materials and Virus Inoculation
Tomato (Solanum lycopersicum L. cv. Moneymaker) (LA2706) were grown in soil in a light-and temperature-controlled chamber at 25 • C with a 16 h light/8 h dark regime. Three-week-old tomato plants were used for mechanical inoculation of ToMV and ToBRFV. ToMV was derived from the infectious clone pTLW3 [13]. For ToBRFV infection we used the Israeli isolate of ToBRFV, ToBRFV-IL (KX619418.1). Plants were dusted with carborundum powder prior to inoculation, and were then rubbed with a phosphate buffer solution (0.01 M, pH 7.0) supplemented with crushed leaves from ToMV-or ToBRFV-infected tomato plants. After three weeks, samples were collected from young leaves showing viral symptoms, distant from the site of inoculation.
For field sample analysis, tomato leaves with mosaic symptoms were collected from two commercial greenhouses in Azriel village, Israel, in December 2020.

CRISPR RNA (crRNA) Design
CRISPR RNA (crRNAs) ( Table 1) were designed using the CRISPOR computational platform for the detection of efficient and specific design of crRNA sequences [47]. Both ToMV and ToBRFV genomes were obtained from the NCBI website (AF332868.1 and KX619418.1, respectively). To create unique crRNAs for each virus, the sequences of both viruses were concatenated into a single long sequence. This single sequence, harboring both viruses, was used as an input for CRISPOR guide gRNA search, allowing the detection of crRNAs that are specific to each virus (Figure 2A). The resulting crRNA sequences (Table 1) were then concatenated to a T7 promoter (T7-crRNA-F; Table 1) sequence and the designed oligonucleotides were ordered from Integrated DNA Technologies (IDT; https://eu.idtdna.com/, Coralville, USA) (T7-crRNA-F).

In Vitro crRNA Transcription
First, each DNA oligo was annealed with oligo T7-crRNA-F. Oligos were suspended in annealing buffer (10 mM Tris pH 8, 50 mM NaCl, 1 mM EDTA) to a final concentration of 100 uM. Next, 10 uL of each oligo was mixed with 10 uL T7-crRNA-F and incubated for 5 min at 95 • C and cooled at room temperature for 1 h. After oligo duplexes were ready, RNA was generated using TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific) according to manufacturer protocol with overnight incubation. Subsequently, RNA was purified using Zymo RNA Clean & Concentrate kit (Zymo), with a DNase I digestion preceding the purification as suggested by the manufacturer's protocol. RNA was diluted 1:150 and measured using a NanoDrop One instrument (Thermo Scientific).

LbCas12a Cleavage Assays
All reactions were prepared on ice. crRNA-LbCas12a complexes were prepared by mixing 62.5 nM gRNA with 50 nM LbCas12a (NEB) in NEBuffer 2.1 (NEB) to a final volume of 20 uL and incubated in 37 • C for 30 min. Next, 1 uM FAM reporter (/56-FAM/TTATTATT/3BHQ_1/)) and 1 nM (120 ng) RT-PCR product were added to the complexes together with 80 uL of 1X concentration NEBuffer 2.1 and incubated for 10 min at 37 • C. Samples were transferred to a black 384 well plate and measured using Tecan Spark plate-reader (Tecan Trading AG, Männedorf, Switzerland) with an excitation wavelength of 485 nm, and emission was measured at 535 nm. Emission was read in relative fluorescence units (RFU) to compare between samples within each experiment.
A reciprocal experiment was performed for ToBRFV detection, only this time with the ToMV RT-PCR product serving as a negative control ( Figure 2D). Here, tobrfv-3 produced a strong fluorescent signal when incubated with the ToBRFV template, which was 6.5 times higher than the ToMV control template ( Figure 2D,E) (n = 4). The tobrfv-5 and tobrfv-9 crRNAs were also efficient in ToBRFV detection, producing fluorescent signals that were 5 and 1.9 higher than the ToMV control ( Figure 2D) (n = 4). These results were consistent in three independent experiments and established the ability of the CRISPR/Cas12a for species-specific identification of ToMV and ToBRFV. As the most efficient detection of ToMV and ToBRFV was achieved by the tomv-1 and tobrfv-3 crRNAs, respectively, these crRNAs were selected for further analysis.

Sensitivity and Specificity of CRISPR/Cas12a-Based Detection
To test the sensitivity of this system, a series of dilutions was performed for each of the ToMV and ToBRFV RT-PCR products ( Figure 3A,B). While 15 ng of RT-PCR product was sufficient for detecting ToMV (Figure 3A), 15-30 ng of RT-PCR product was needed for the detection of ToBRFV ( Figure 3B). As 15-30 ng of PCR products was the minimal threshold for detection, we further used these parameters to examine if the CRISPR/Cas12a system could specifically detect each virus in the case of a mixture of ToMV and ToBRFV. To test this, detection assays were performed individually using tomv-1 or tobrfv-3 on a mixture of RT-PCR products from both ToMV and ToBRFV ( Figure 3C,D). Notably, 15 ng of ToMV was detected in a mixture with 15 ng of ToBRFV, resulting in a fluorescent signal that was 85% higher than the signal obtained with 30 ng of ToBRFV only ( Figure 3C). In addition, the CRISPR/Cas12a system was also able to detect 15 ng of ToBRFV mixed with 15 ng of ToMV, producing a fluorescent signal that was 3.5-fold higher than the signal received with only 30 ng of ToMV ( Figure 3D). These results suggest that the CRISPR/Cas12a system can be used to detect ToBRFV, even in the case of mixed infection with the related virus ToMV.

CRISPR/Cas12a-Based Detection of ToBRFV from Field Samples
Next, we tested the applicability of the CRISPR/Cas12a system for specific detection of tobamoviruses in two field samples from Azriel village, Israel. In two separate greenhouses, plants with severe mosaic and shoestring leaf symptoms were detected, consistent with tobamovirus infection ( Figure 4A). We used our CRISPR/Cas12a system to diagnose if the plants were infected with ToMV or ToBRFV. Analysis using ToMV crRNA tomv-1 revealed that both tomato plants were likely not infected with ToMV, as indicated by the weak fluorescent signal that was similar to the ToBRFV as negative control ( Figure 4B). In marked contrast, the ToBRFV crRNA tobrfv-3 showed high fluorescence for both samples, which was similar to the ToBRFV positive control ( Figure 4C). To confirm these results, RT-PCR products from both samples were sequenced. Indeed, both samples showed 100% identity to ToBRFV-IL (KX619418.1) and 76.91% to ToMV (AF332868). These results establish that the CRISPR/Cas12a system can detect ToBRFV in field samples.
higher than the signal received with only 30 ng of ToMV ( Figure 3D). These results suggest that the CRISPR/Cas12a system can be used to detect ToBRFV, even in the case of mixed infection with the related virus ToMV.

CRISPR/Cas12a-Based Detection of ToBRFV from Field Samples
Next, we tested the applicability of the CRISPR/Cas12a system for specific detection of tobamoviruses in two field samples from Azriel village, Israel. In two separate greenhouses, plants with severe mosaic and shoestring leaf symptoms were detected, consistent with tobamovirus infection ( Figure 4A). We used our CRISPR/Cas12a system to diagnose if the plants were infected with ToMV or ToBRFV. Analysis using ToMV crRNA tomv-1 revealed that both tomato plants were likely not infected with ToMV, as indicated by the weak fluorescent signal that was similar to the ToBRFV as negative control ( Figure 4B). In marked contrast, the ToBRFV crRNA tobrfv-3 showed high fluorescence for both samples, which was similar to the ToBRFV positive control ( Figure 4C). To confirm these results, RT-PCR products from both samples were sequenced. Indeed, both samples showed 100% identity to ToBRFV-IL (KX619418.1) and 76.91% to ToMV (AF332868). These results establish that the CRISPR/Cas12a system can detect ToBRFV in field samples.

Discussion
ToBRFV is an emerging tobamovirus, which is spreading rapidly throughout the world and poses a substantial threat to the global tomato industry. ToBRFV symptoms

Discussion
ToBRFV is an emerging tobamovirus, which is spreading rapidly throughout the world and poses a substantial threat to the global tomato industry. ToBRFV symptoms are similar to other tobamoviruses, such as ToMV. These challenges drove the development of new real-time PCR and LAMP assays for specific ToBRFV detection [31,48]. While it was shown that CRISPR/Cas12a can be used to detect plant viruses [44], it was unknown if it could be applied to distinguish between closely related plant viruses of the same genus. Here, we provide evidence that the CRISPR/Cas12a technology can also be applied to identify ToBRFV and distinguish it from the closely related ToMV (Figure 2). Using this method, ToBRFV can be detected with low concentrations of RT-PCR products, and also when mixed with ToMV ( Figure 3). Detection of ToBRFV in field samples ( Figure 4) demonstrates this technology's potential as a bona fide virus identification method that can be used in agricultural practice.
Further research is required to determine if our method is able to distinguish ToBRFV from solanaceous-infecting viruses other than ToMV. The tobrfv-3 crRNA sequence is highly specific to ToBRFV, and shows no significant similarity to the closely related viruses TMV (V01408), tomato mottle mosaic virus (ToMMV) (KF477193) and rehmannia mosaic virus (RheMV) (EF375551) according to BLAST analysis. Therefore, it can be anticipated that this CRISPR/Cas12a system will also be able to distinguish between ToBRFV and these tobamoviruses.
While the current setting of our experiment still requires a thermocycler, further development can enhance the CRISPR/Cas12a method for more accessible and user-friendly applications. For example, CRISPR/Cas12a can be combined with isothermal amplification methods such as loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA), to detect ToBRFV quickly and accurately in less than 15 min. Such detection platforms named SHERLOCK and DETECTR [42,49] were successfully commercialized for SARS-Cov-2 detection based on CRISPR/Cas13 and Cas12a, respectively. Importantly, such technology can be applied using simple incubation instrumentation, such as a low-cost hand warmer (~USA Dollars 0.3), rather than expensive and complex thermocyclers [50]. Moreover, the LAMP/RPA-coupled CRISPR-Cas12 technology is cost effective, with a price of 2-5 USD/reaction [51], which is comparable to existing RT-PCR and real-time RT-PCR techniques. Our protocol still relies on the RT-PCR step. Replacement of RT-PCR with isothermal amplification such as RPA or LAMP, as in SHERLOCK and DETECTR, would significantly improve the applicability of our system and enable its future application in the field.
Recently, it was shown that the amplification step may be avoided through signal amplification. One such option is to use gold nanoparticles, or L-methionine gold nanoclusters combined with electrochemiluminescence to enhance and stabilize the signal [52,53]. Another approach was recently reported where the use of several crRNAs, targeting several loci within the target DNA, significantly increased the fluorescent signal achieved following Cas13 activation [54]. In addition, several reports have shown that the output signal can be visual rather than fluorescent, further simplifying the detection procedure. Such outputs may be colorimetric, or by using lateral flow strips, similar to common pregnancy tests [42,52]. Collectively, these approaches will allow onsite, rapid, and accurate detection without the need for specialized training or equipment. These techniques will be especially relevant for the detection of closely related pathogen species or strains, and resistance-breaking variants.