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. 2023 Mar 1;228:115179. doi: 10.1016/j.bios.2023.115179

An ultra-sensitive one-pot RNA-templated DNA ligation rolling circle amplification-assisted CRISPR/Cas12a detector assay for rapid detection of SARS-CoV-2

Zaobing Zhu a, Yongkun Guo a, Chen Wang a, Zifeng Yang b, Rong Li a, Zhiqi Zeng b, Hui Li c, Dabing Zhang a, Litao Yang a,∗
PMCID: PMC9974209  PMID: 36878066

Abstract

Rapid, sensitive, and one-pot diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an extremely important role in point-of-care testing (POCT). Herein, we report an ultra-sensitive and rapid one-pot enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay, termed OPERATOR. OPERATOR employs a single well-designed single-strand padlock DNA, containing a protospacer adjacent motif (PAM) site and a sequence complementary to the target RNA which procedure converts and amplifies genomic RNA to DNA by RNA-templated DNA ligation and multiply-primed rolling circle amplification (MRCA). The MRCA amplicon of single-stranded DNA is cleaved by the FnCas12a/crRNA complex and detected via a fluorescence reader or lateral flow strip. OPERATOR presents outstanding advantages including ultra-sensitivity (1.625 copies per reaction), high specificity (100%), rapid reaction speed (∼30 min), easy operation, low cost, and on-spot visualization. Furthermore, we established a POCT platform by combining OPERATOR with rapid RNA release and a lateral flow strip without professional equipment. The high performance of OPERATOR in SARS-CoV-2 tests was confirmed using both reference materials and clinical samples, and the results suggest that is readily adaptable for point-of-care testing of other RNA viruses.

Keywords: OPERATOR, SARS-CoV-2, CRISPR/Cas12a, RCA, RNA-Templated DNA ligation

1. Introduction

A novel coronavirus, termed SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), was first reported in December 2019, in Wuhan, China (Zhu et al., 2020). In the past two years, the virus had infected more than hundreds of millions of people worldwide. So far, the pandemic has not been effectively contained and continues to place a huge burden on global public health and socio-economic activities (Asare Vitenu-Sackey and Barfi, 2021; Laborde et al., 2020; Nicola et al., 2020).

Currently, there are two main methods for clinical diagnosis of SARS-CoV-2; one is reverse-transcription quantitative PCR (RT-qPCR) based on a nucleic acid amplification (NAA), and the other is rapid antigen detection (RAD) based on serological analysis (Whitman et al., 2020). RT-qPCR, a routine laboratory method, is considered the gold standard worldwide for the early detection of SARS-CoV-2 (Pokhrel et al., 2020). RT-qPCR has obvious advantages including high specificity and sensitivity. However, dependence on an expensive thermal cycler instrument, an experienced operator, high-quality extracted RNAs, and long test duration (>1.5 h) limit its application in point-of-care testing (POCT) (Al-Tawfiq and Memish, 2020; Dong et al., 2020; Suo et al., 2020; Wu and McGoogan, 2020). RAD tests for the SARS-CoV-2 antigen using immobilized SARS-CoV-2 antibody coated on a lateral flow strip (LFS), and advantages include on-spot visualization, rapid test duration, low cost, and instrument-independent operation. However, RAD tests also have limitations including low sensitivity, high false positive rate, and false negatives (Döhla et al., 2020). Therefore, the development of a rapid, specific, and sensitive POCT method targeting nucleic acids is urgently needed, but this remains challenging.

The isothermal nucleic acid amplification (iNAA) test, a methodology for amplifying DNA/RNA templates at a single temperature, is often used to develop POCT approaches such as rolling cycle amplification (RCA), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA) (Yan et al., 2014). Recently, clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated (Cas), a component of an adaptive immune system in bacteria and archaea that defends against infectious nucleic acids, can be used for nucleic acid detection as well as gene editing (Liu et al., 2022, van Dongen et al., 2020; Wang et al., 2020). Cas proteins can accurately target any region of DNA or RNA in association with a single guide RNA (CRISPR RNA, crRNA) that matches the target DNA or RNA (Makarova et al., 2020; Zetsche et al., 2015). In particular, promiscuous trans-cleavage activity triggered by specific target recognition and cleavage was discovered, which enables Cas proteins (e.g., Cas12a, Cas12b, and Cas13a) to be used as biosensors for DNA/RNA readout in POCT (Chen et al., 2018; Gootenberg et al., 2017; Li et al., 2018b). Several CRISPR/Cas-mediated isothermal methods have been developed for the detection of RNA and DNA viruses, such as SHERLOCK, HOLMES, and DETECTOR (Jia et al., 2020; Strich et al., 2019; Yin et al., 2021). Among these methods, two iNAA techniques (RT-RPA and RT-LAMP) were mainly used to associate with various CRISPR/Cas biosensors (Cas9, Cas12a, and Cas13a), including RT-RPA-Cas9 (Azhar et al., 2021; Marsic et al., 2021; Xiong et al., 2021), RT-RPA-Cas12a (Aman et al., 2021), RT-LAMP-Cas12a (Ali et al., 2020; Broughton et al., 2020) and RT-RPA-Cas13a (Arizti-Sanz et al., 2020). Compared with LAMP and RPA, RCA isothermally amplifies the circular DNA template with phi 29 DNA polymerase, producing long single-stranded DNA (ssDNA) with circular template sequence repeats in tandem. This long ssDNA amplicon can be detected by DNA hybridization or a fluorescence dye (Takahashi et al., 2018). However, the application of RCA in iNAA is limited due to the challenge of accurate and specific monitoring for generated ssDNA amplicons.

Following the emergence of a novel coronavirus, various isothermal assays for SARS-CoV-2 have been established by combining iNAA and CRISPR/Cas-based biosensors, such as miSHERLOCK (de Puig et al., 2021), DETECTR (Broughton et al., 2020), SHINE (Arizti-Sanz et al., 2020), AIOD-CRISPR (Ding et al., 2020), POC-CRISPR (Chen et al., 2021), Cas-HCR (Yang et al., 2021), and iSCAN (Ali et al., 2020). Most of these methods require multiple operation steps due to multiple reaction temperatures (e.g., 42 °C for reverse transcription, 60−65 °C or 37−42 °C for amplification, 37 °C for cleavage), complex reagent composition with multi-reactive enzymes (i.e., reverse transcription polymerase, DNA polymerase, Cas protein), and relatively long detection duration (∼60 min). These shortcomings limit the potential application of POCT. In addition, reverse transcription is still a necessary step for regular RNA detection methods, and very few transcription-free methods have been reported for RNA detection except for RCA-assisted CRISPR assay (Shi et al., 2022; Wang, R. et al., 2020). In the RCA-assisted CRISPR assay, three reactions of padlock probe ligation, RCA, and CRISPR/Cas cleavage were performed step-wise with a longer reaction duration time (>6 h). The integration of multiple-step reactions is still challenging.

In the present study, we report a novel reverse transcription-free one-pot enzyme-catalyzed RCA-assisted CRISPR/FnCas12a detector assay for coronavirus SARS-CoV-2 detection, termed OPERATOR, which integrates RNA-templated padlock probe ligation, multiply-primed RCA (MRCA), and CRISPR/FnCas12a cleavage into a one-pot reaction. In OPERATOR, the target RNA is converted to a circular ssDNA by RNA-templated padlock probe ligation, the cyclized padlock probe is amplified as the template in RCA using a single universal multiply-primer, and RCA amplicons are recognized by the FnCas12a/crRNA complex and detected by a fluorescence signal reader or LFS. OPERATOR can accurately detect SARS-CoV-2 virus RNA with highly sensitive and rapid reaction speed.

2. Materials and methods

2.1. Materials

Certified reference materials (CRMs) for SARS-CoV-2 (GBW (E) 019089) and reference material for 2019-nCoV pseudo virus RNA (NIM-RM5203) were purchased from the National Centre for Reference Materials (NCRM), Beijing, China. Seven RMs for different respiratory viruses were kindly supplied by the Shanghai Institute of Measurement and Testing Technology (SIMT), Shanghai, China. Details for all RMs are listed in Supplemental Table S1. RNA was extracted from virus samples using a QIAamp Viral RNA Kit (Qiagen, Hilden, Germany), and then reverse transcribed into cDNA using FastKing RT Kit (Tiangen, Beijing, China). The quality and quantity of extracted RNA were evaluated using a NanoDrop 2000 UV/vis spectrophotometer (NanoDrop Technologies, LLC, Wilmington, DE, USA). The ssDNA, double-stranded DNA (dsDNA), and RNA templates of partial SARS-CoV-2 E genes used for padlock probe cyclization are described in Supplemental File 1.

2.2. Design and synthesis of padlock probes, crRNAs, primers, and probes

Based on a previous report, an ideal Padlock probe for generating a circular ssDNA should be < 100 nucleotides in length, the arms at both ends should be < 20 nucleotides, and the linker between the two arms should be longer than the sum of arms (Takahashi et al., 2018). A total of six padlock probes containing various linker regions were designed targeting the E gene of SARS-CoV-2 (Gene ID: 43740570) using OligoAnalyzer 3.1 (https://sg.idtdna.com/calc/analyzer) and RNAfold (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi). The structure diagram of the designed padlock probes is shown in Supplemental Fig. S1. Probes comprised a left arm, a PAM site, a linker, and a right arm in tandem. The PAM site of TTT was artificially introduced into the padlock probe to eliminate the dependence of the PAM motif on in vitro CRISPR/FnCas12a cleavage.

The in vitro transcription templates were prepared by annealing the synthesised oligonucleotides with crRNA-F (Supplemental Table S2), following the same procedures as previously described (Li et al., 2016). Subsequently, in vitro transcription was performed using a HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, USA). Transcribed RNAs were purified using RNA Clean & Concentrator TM-100 (ZYMO RESEARCH, Irvine, USA) and stored at −70 °C for further experiments. The quality and quantity of extracted RNA were evaluated using a NanoDrop 2000 UV/vis spectrophotometer (NanoDrop Technologies, LLC, Wilmington, DE, USA).

A universal thiophosphate-modified random hexamer (5′-NpNpNpNpNpsNs-3′, 6Ns) with two sulfur-modified bases at the 3′ end was used for multiply-primed RCA (Dean et al., 2001). Two different types of fluorescence-labeled DNA reporter probes were designed; a cis-probe (CP) that hybridizes with the RCA product, generates a dsDNA recognition region for the FnCas12a/crRNA complex, and monitors RCA products using a fluorescence signal; and a trans-probe (TP or LFP) for trans cleavage by CRISPR/FnCas12a and measurement of the RCA product by TP or LFP. The TP was used for fluorescence inspection, and the LFP was used for visual inspection of the OPERATOR coupled with LFS. All padlock probes, crRNAs, primers, and probes are listed in Supplemental Table S2 and were synthesised by Invitrogen Company (Shanghai, China).

2.3. DNA/RNA-templated DNA ligation

DNA-templated DNA ligation and RNA-templated DNA ligation were performed to create the circular ssDNA. Both RNA- and DNA-templated DNA ligation were performed in a volume of 20 μL containing 2 μL of T4 DNA ligase (5 U/μL; Thermo Fisher, USA), 2 μL 10 × ligation buffer (Thermo Fisher), 1 μL PEG 4000, 1 μL padlock probe (10 pmol), 1 μL RNA or DNA template (∼10 pmol) and 13 μL DEPC water (LABTOP BIO, Shanghai, China). The ssDNA or RNA-template DNA ligation reaction was incubated at 37 °C for 30 min. In the dsDNA-template DNA ligation reaction, the dsDNA and padlock probe were pre-incubated at 95 °C for 5 min before adding T4 DNA ligase. The ligation products were analyzed by 2% (w/v) agarose gel electrophoresis with GelRed staining.

2.4. Multiply-primed rolling circle amplification

Multiply-primed rolling circle amplification (MRCA) was performed using the 6Ns thiophosphate-modified random hexamer. The MRCA reaction was conducted in a 20 μL volume containing 10 μL 2 × RCA buffer (100 mM Tris-HCl, 20 mM MgCl2, 8 mM DTT, 20 mM (NH4)2SO4, 4 mM dNTP, 2 mg/mL BSA), 1 μL Phi29 DNA polymerase (10 U/μL; New England Biolabs), 1 μL 6Ns primer (10 μM final concentration), 1 μL ligation product and 7 μL DEPC water (LABTOP BIO, Shanghai, China). The MRCA reaction was incubated at 37 °C for 1 h and denatured at 65 °C for 10 min. MRCA products were analyzed by 2% (w/v) agarose gel electrophoresis with GelRed staining.

2.5. In-vitro CRISPR/FnCas12a cleavage and readout of results

The FnCas12a protein was expressed and purified according to our previous research (Zhu et al., 2022). The in vitro CRISPR/FnCas12a cleavage reaction was optimized in a total volume of 20 μL containing 2 μL 10 × NEBuffer 2.1 (New England Biolabs), 1 μL FnCas12a (250 nM final concentration), 1 μL crRNA (250 nM final concentration), 1 μL RCA product and 15 μL ddH2O. The in vitro cleavage reaction was performed at 37 °C for 30 min and cleaved products were analyzed by 2% agarose gel electrophoresis with GelRed staining.

Fluorescence or visual inspection of cleaved products was performed by adding additional fluorescent probes into CRISPR/FnCas12a cleavage reactions. Each reaction was prepared in a volume of 20 μL containing 2 μL 10 × NEBuffer 2.1 (New England Biolabs), 1 μL FnCas12a (250 nM, final), 1 μL crRNA (250 nM, final), 1 μL MRCA product, 1 μL cis-probe (200 nM, final), 1 μL trans-probe (200 nM, final) and 13 μL ddH2O. For fluorescence detection, the cleavage reaction was carried out on a 7900 HT Fast Real-Time RCR system (Applied Biosystems, CA, USA) at 37 °C for 60 min, and the fluorescent signal was monitored every 1 min. Data analysis was performed by GraphPad Prism 9 software (GraphPad Software, San Diego, USA). For visual inspection, cleaved products were visualized using the LFS developed in our lab (Zhu et al., 2022).

2.6. OPERATOR reaction

Each OPERATOR reaction was performed in a volume of 20 μL containing 10 μL 2 × OPERATOR buffer (80 mM Tris-HCl, 20 mM MgCl2, 20 mM DTT, 4 mM dNTPs, 2 mg/mL BSA, 1 mM ATP), 1 μL 6Ns primer (10 μM final concentration), 2 μL T4 DNA ligase (5 U/μL), 1 μL FnCas12a (250 nM), 1 μL crRNA (250 nM, final), 1 μL padlock probe (500 nM, final), 1 μL cis-probe (200 nM, final), 1 μL trans-probe (200 nM, final), 1 μL RNA template and 2 μL DEPC water (LABTOP BIO, Shanghai, China). Each OPERATOR reaction was incubated at 37 °C for 40–60 min. For fluorescence signal detection, OPERATOR was carried out on a 7900 HT Fast Real-Time PCR system (Applied Biosystems, CA, USA). For visual inspection, OPERATOR products were visualized using LFS.

2.7. Reverse transcription quantitative real-time PCR (RT-qPCR)

RT-qPCR analysis of clinical samples was performed using a commercial SARS-Cov-2 kit (Zhuhai Huirui Biotech Company, Zhuhai, China). Each RT-qPCR reaction was prepared in a volume of 20 μL containing 10 μL qPCR master mix, 1 μL RNA template, and 7 μL ddH2O. RT-qPCR analysis was carried out on a 7900HT Fast Real-Time PCR system (Applied Biosystems, CA, USA) at 50 °C for 10 min, 95 °C for 5 min, then 40 cycles at 95 °C for 15 s, and 60 °C for 45 s. The fluorescence signal was monitored at 60 °C. Each reaction was performed in triplicate, with three biological replicates.

2.8. Evaluation of SARS-CoV-2 OPERATOR assay performance

OPERATOR performance was evaluated in terms of specificity, sensitivity, and clinical sample detection. Seven respiratory viruses RM RNAs, namely SARS-CoV-2 (2019n-CoV), Parainfluenza virus type 1, Influenza A virus (H7N9), Influenza A virus (H1N1), Influenza B virus (Victoria), SARS-CoV, and Middle East respiratory syndrome-related coronavirus (MERS-CoV) were used to evaluate the specificity of the developed OPERATOR assay targeting the E gene of SARS-Cov-2. To assess sensitivity, a series of RNA dilutions of SARS-Cov-2 virus CRM (GBW [E] 019089), corresponding to concentrations of 162,500, 16,250, 1,625, 162.5, 16.25, 1.625, 0.50 and 0.25 copies/μL were prepared and tested. The limit of detection of 95% (LOD) was determined via probit analysis based on 20 replicates of each dilution (van Kasteren et al., 2020). A total of 110 RNAs from clinical samples (coded from S1 to S110) were tested in OPERATOR assay. Each sample was tested in triplicate.

3. Results

3.1. OPERATOR principle and design

In this study, we designed a novel, ultrasensitive, rapid, one-pot isothermal method to detect SARS-CoV-2, termed OPERATOR, which is based on RNA-templated padlock probe ligation, MRCA, and CRISPR/FnCas12a in vitro detection. OPERATOR is the first approach to integrate padlock probe ligation, MRCA, and CRISPR/FnCas12a cleavage into a single-tube reaction. The design and principle are shown in Fig. 1 a. The whole OPERATOR procedure involves three steps: RNA-templated padlock probe ligation (Step 1), MRCA (Step 2), and in vitro CRISPR/FnCas12a cleavage (Step 3). Specifically, a designed padlock DNA probe, containing a PAM motif and sequences complementary to the target RNA of the virus, hybridizes with the target RNA template to trigger ligation of the 5′ and 3′ termini of the padlock probe by high-fidelity T4 DNA ligase. The resulting single-stranded circular padlock probe functions as a template in a multiply-primed RCA reaction with a universal random hexamer primer (6Ns), in which ssDNA amplicons containing repetitive targets in tandem are reacted in the presence of Phi29 DNA polymerase. After the RCA reaction, the cis-probe complementary to the target gene sequence is added, and it hybridizes with the repetitive target regions of ssDNA RCA amplicons to form a dsDNA assembly. The FnCas12a/crRNA complex recognizes the introduced PAM domain (TTT) within the dsDNA assembly and specifically cleaves the target dsDNA region, and fluorescence is emitted from the digested cis-probe. At the same moment, nonspecific ssDNA trans-cleavage activity of FnCas12a is triggered, which cut the trans-probe into pieces and emits a fluorescence signal. Fluorescence from the cleaved cis-probe and trans-probe is measured by a fluorescence reader or LFS.

Fig. 1.

Fig. 1

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The principle and workflow of OPERATOR. (a) Working principle. Step 1, RNA-templated DNA ligation; Step 2, Multiply-primed rolling cycle amplification (MRCA) Step 3, in vitro CRISPR/FnCas12a cleavage. (b) Schematic diagram showing the workflow of step-wise reactions for (1) and OPERATOR (2).

The one-pot reaction of OPERATOR successfully integrates the above three steps for POCT, which simplifies all operations and reduces the reaction duration. Fig. 1b shows the workflow of step-wise reactions and the one-pot reaction of OPERATOR, respectively. In the step-wise approach, three reactions are performed step by step with multiple pipetting, solution transfer, and long reaction duration. By contrast, in a one-pot reaction, only the addition of an RNA template into the prepared one-pot reaction mixture is needed without additional pipetting and solution transfer, and the reaction duration could be reduced dramatically.

3.2. The padlock probe E-PL-1 can be cyclized efficiently based on the target RNA sequence

The design of the padlock probe is critical because it determines the efficiency of padlock circle creation and subsequent RCA. The padlock probe is designed to contain four parts from the 5′ to the 3′ ends; a left arm, a linker, a PAM motif, and a right arm (Fig. 2 a). The sequences of both arms should perfectly complement the interest sequence of the target RNA to ensure specific hybridization with the target RNA and cyclization. The PAM site of FnCas12a recognition must be introduced into the padlock probe to eliminate sequence independence in crRNA design. Also, the complex secondary structures should be avoided in the RNA-DNA hybridization region to ensure the high sealing efficiency of the padlock probe without annealing. Six padlock probes (E-PL-1 to E-PL-6) were designed with various linker sequences and evaluated in RNA-templated DNA ligation and MRCA reactions (Supplemental Fig. S1). Agarose gel electrophoresis analysis of ligation and RCA products showed that the padlock probe circle was successfully created using E-PL-1 and E-PL-6, and effectively amplified as the template in RCA (Supplemental Fig. S2). However, fluorescence analysis of in vitro CRISPR/FnCas12a cleavage of the RCA products revealed an obvious fluorescence signal only with E-PL-1 (Fig. 2b). These results indicate that the designed E-PL-1 padlock probe is suitable for creating the ssDNA circle, and subsequent RCA and CRISPR/FnCas12a cleavage reactions. Therefore, the linker sequence in the E-PL-1 padlock probe was used for all subsequent padlock probe designs for further experiments.

Fig. 2.

Fig. 2

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Feasibility of the enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay in step-wise reactions. (a) Conceptual diagram of a padlock probe and PAM-free design. (b) Fluorescence signal readout of the assays using various PLs. (c) Agarose gel electrophoresis analysis of CRISPR/FnCas12a in vitro cleavage of RCA amplicons. (d) Fluorescence signal readout of CRISPR/FnCas12a in vitro cleavage of RCA amplicons. DP, CP, and TP are reactions with dual probes (trans- and cis-probes), cis-probe, and trans-probe, respectively. Cas12 (−) and crRNA (−) are reactions without Cas12a and crRNA, respectively. N is the reaction without the RCA product.

3.3. The feasibility of OPERATOR for RNA analysis is proved by step-wise reactions

Since previous reports only confirmed that the sealing efficiency of DNA padlock probes is high with ssDNA (Yue et al., 2021), we first evaluated the sealing efficiency of RNA-templated padlock DNA ligation compared with using ssDNA and dsDNA templates. Our results showed that the ssDNA circle of the padlock probe was created in all reactions with different templates, and large quantities of RCA products were produced in all reactions, indicating that RNA-templated DNA ligation can efficiently create circular ssDNA padlock probes, although ssDNA-templated ligation presents slightly higher sealing efficiency (Supplemental Fig. S3a). To improve the efficiency of amplification, we adopted a multiply-primer strategy according to the previous report (Dean et al., 2001; Yue et al., 2021). Our results showed that the amplification efficiency and yield of MRCA were much higher than RCA, and the optimal primer concentration was determined to be 10 μM (Supplemental Figs. S3b–3c).

In previous work, the FnCas12a/crRNA complex was mainly used to cleave the dsDNA template and trigger trans-cleavage activity for fluorescence detection, although FnCas12a is reportedly capable of ssDNA cleavage independent of the PAM motif, and triggering trans-cleavage activity (Li et al., 2018a; Swarts and Jinek, 2019). In our work, we first designed and used FnCas12a to identify and assess ssDNA by introducing an additional fluorescence reporter probe (cis-probe) into the CRISPR/FnCas12a cleavage reaction. The results confirmed that the RCA products could be recognized and cleaved into short ssDNA fragments by the FnCas12a/crRNA complex (Fig. 2c), consistent with previous reports (Li et al., 2018a), indicating that FnCas12a can be used to identify the ssDNA directly.

Next, we designed the cis-probe the match the ssDNA of the RCA amplicon to form a dsDNA region, dsDNA is then cleaved by the FnCas12a/crRNA complex, and the fluorescence signal is emitted from the digested cis-probe and trans-probe simultaneously. Our results showed that the highest fluorescence intensity was achieved with two probes, and the fluorescence intensity was twice that of the CRISPR/FnCas12a cleavage reaction with only cis-probe or trans-probe (Fig. 2d). The double fluorescence mechanism can effectively improve the fluorescence intensity, which is conducive to improving the sensitivity when detecting trace amounts of ssDNA products. These results preliminarily proved the feasibility of the MRCA and FnCas12a detection approach for RNA molecule analysis.

3.4. Establishing and optimizing single-tube OPERATOR

In the development and application of both laboratory and on-spot nucleic acid detection methods, simple reaction steps and operations are preferable. However, most reported methods employing CRISPR/Cas in identification or reporter systems involve multiple steps, such as target amplification, amplicon identification, and analysis of cleaved products (Broughton et al., 2020; Chen et al., 2021; Gootenberg et al., 2017; Li et al., 2018b). In this work, we tried to establish a one-pot process by combining three-step reactions of OPERATOR into a single tube. To further explore the potential for the development of OPERATOR reaction, we evaluated the temperature and reaction duration of RNA-templated DNA ligation, because T4 DNA ligase presents high activity over a wide temperature range (16°C–42 °C). The padlock probe could be cyclized at various temperatures ranging from 16 °C to 42 °C and amplification were effective in the RCA reaction (Supplemental Fig. S4a). Fluorescence analysis results further showed that ligation efficiency from 16 °C was slightly higher than other temperatures (Fig. 3 a). Considering that the lower temperature (16 °C) will severely reduce the amplification efficiency of Phi29 and the cleavage efficiency of FnCas12a (Malzahn et al., 2019), we selected 37 °C as a candidate temperature for further integrating the reactions of ligation, MRCA, and CRISPR/Cas12a cleavage into one single tube. The padlock probe could be cyclized with a reaction duration ranging from 5 min to 60 min (Fig. 3b, Supplemental Fig. S4b), indicating that ligation duration is not a significant factor, and 5 min is sufficient. The results of reaction temperature and reaction time evaluation demonstrated the possibility of combining the three reactions into a one-pot process.

Fig. 3.

Fig. 3

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Optimization and development of an OPERATOR procedure. (a) Fluorescence signal readout of the MRCA reactions using ligated products from different reaction temperatures as templates. (b) Fluorescence signal readout of MRCA reactions using ligated products from different reaction durations as templates. (c) Fluorescence signal readout of MRCA reactions using ligated products from different buffers as templates. (d) Fluorescence signal readout of MRCA amplification reactions using different buffers. (e) Fluorescence signal readout of ligation-mediated RCA using buffers B1, B2, and B3. (f) Fluorescence detection of CRISPR/FnCas12a cleavage reactions with various buffers of B1 to B8. (g) Fluorescence detection of CRISPR/FnCas12a cleavage reactions employing ligation-mediated RCA products at different reaction durations (10 min–80 min) as templates. (h) Fluorescence signal detection of OPERATOR with optimized one-pot reaction.

We considered the activity characteristics of various enzymes and focused on reaction buffer optimization. Eight reaction buffers (B1 to B8) were prepared (Supplemental Table S3), and the suitability of the prepared buffers for one pot was tested. Firstly, the eight buffers were tested in RNA-template DNA ligation, and the cyclized padlock probe was amplified in multiply-primed RCA. Agarose gel electrophoresis analysis of RCA products showed obvious RCA amplicons in reaction with the cyclized padlock probe with B1–B3 and B5–B8 (Supplemental Fig. S5), indicating that most buffers could be used for RNA-template DNA ligation. Further fluorescence inspection of CRISPR/FnCas12a cleavage of RCA products showed that reactions with B1, B2, and B5 presented higher fluorescence intensity than those with B3 and B6–B8 and almost no fluorescence was observed in the reaction with B4 (Fig. 3c). Thus, RNA-template DNA ligation with B1, B2, and B5 presented better cyclization efficiency.

Next, eight buffers were tested in MRCA with the same cyclized padlock probe. Agarose gel electrophoresis analysis showed that large quantities of RCA amplicons were generated in reactions with B1–B3, but quantities were much lower for B5 and B6. Reactions with B2 and B3 produced the most RCA amplicons (Supplemental Fig. S6). Fluorescence analysis of CRISPR/FnCas12a cleavage also confirmed that B1–B3 and B5 and B6 could support efficient RCA amplification; B2 displayed the best amplification efficiency, followed by B3, B1, B6, and B5 (Fig. 3d). Considering that B4 performed poorly in the ligation reaction and that B5–B8 was inefficient in the amplification step, B1–B3 was selected for further evaluation.

We then combined RNA-templated DNA ligation and MRCA into a single-tube reaction with B1–B3, according to the results of the previous two steps. Large quantities of RCA products were produced using all three buffers (Supplemental Fig. S7). Fluorescence analysis showed that reactions with B1 and B2 produced much higher fluorescence intensity (Fig. 3e). Therefore, to determine the optimal buffer for one pot, we tested the shearing efficiency of the CRISPR/FnCas12a with eight buffers. Reaction with B2 is second only to B6 (Fig. 3f), however, considering the low efficiency of B6 ligation and amplification, we finally choose B2 as the candidate buffer to integrate CRISPR/FnCas12a cleavage and the combined ligation/MRCA reactions.

Finally, we tested the duration of ligation/MRCA in B2, and a fluorescence signal was detected within 20–60 min (Fig. 3g). Finally, a single-tube OPERATOR combining all three steps (ligation, MRCA, and CRISPR/FnCas12a cleavage) was established using buffer B2, an obvious fluorescence signal was observed, and the signal could be detected after 20 min (Fig. 3h).

3.5. OPERATOR analysis of SARS-CoV-2 is highly specific, ultrasensitive, and amenable to clinical samples

The padlock probe and crRNA were designed to target the E gene of SARS-CoV-2 to develop the OPERATOR assay. The sequence of the target region of the E gene is theoretically highly specific (Fig. 4 a). OPERATOR assay was successfully established using the designed padlock probe and crRNA.

Fig. 4.

Fig. 4

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Performance evaluation of OPERATOR assays for SARS-Cov-2 detection. (a) Alignment of the target sequence of interest of the E gene of SARS-CoV, MERS-CoV, and SARS-CoV-2. (b) Heatmap of orthogonal specificity verification among the three OPERATOR assays for SARS-CoV, MERS-CoV, and SARS-CoV-2. (c) Specificity evaluation of the OPERATOR assay for SARS-CoV-2 employing different respiratory virus RNAs (RMs-1 to RMs-7) as templates. (d) Sensitivity evaluation of the OPERATOR assay for SARS-CoV-2 employing a series of RNA dilutions as the templates. (e) Fluorescence intensity and CT value of clinical samples (S1 to S110) using the OPERATOR and RT-qPCR assay.

To evaluate the specificity of the developed OPERATOR assay, an orthogonal test was first performed employing the developed OPERATOR assays for three viruses (SARS-CoV, SARS-CoV-2, and MERS-CoV). A fluorescence signal could only be observed in each assay with the corresponding virus (Fig. 4b). Secondly, in the SARS-CoV-2 OPERATOR reactions employing seven different respiratory disease viruses as the templates, an obvious fluorescence signal was only observed in the reaction with SARS-CoV-2 (Fig. 4c). The results from the two validation experiments confirmed the high specificity of the developed OPERATOR assay for SARS-CoV-2.

The sensitivity of SARS-CoV-2 assays was evaluated using gradient dilutions (162,500, 16,250, 1,625, 162.5, 16.25, 1.625, 0.50, and 0.25 copies/μL) of CRM (GBW [E] 019089) as calibrators. In OPERATOR, the obvious fluorescence signal was observed in all reactions except for the reaction with the dilutions of 0.25 and 0.5 copies/μL (Fig. 4d), and the LOD was determined to be as low as 1.625 copies per OPERATOR reaction at the confidence of 95%. The constructed standard curves between LG (concentration) and fluorescence signal intensity for OPERATOR assays revealed high linearity, with values of 0.9328 (Supplemental Fig. S8). Sensitivity evaluation proved that the developed OPERATOR assay was ultrasensitive compared with previously reported methods (Table 1 ).

Table 1.

Comparison of OPERATOR with existing detection techniques.

Methods Operation steps Specificity (Resolution) Sensitivity Duration PAM-free One-pot Reference
SHINE (1) RT-RPA 100% 10 copies/μL ∼1.3 h Yes No Arizti-Sanz et al. (2020)
(2) CRISPR/Cas fluorescence assay/LFS
DETECTR (1) LAMP 100% 10 copies/μL <40 min No No Broughton et al. (2020)
(2) CRISPR/Cas fluorescence assay/LFS assay
RACE (1) RT-RPA High 90-100 fM (∼5.4 copies/μL) >2 h Yes No Wang, R. et al., 2020
(2) CRISPR/Cas LFS assay
POC-CRISPR (1) RT-RPA High 1 genome/100 μL ∼30 min No No Chen et al. (2021)
(2) CRISPR/Cas LFS assay
Cas-HCR Cas13 and HCR circuits 100% 10 aM (∼6 copies/μL) ∼1 h NA Yes Yang et al. (2021)
opvCRISPR (1) One-pot reaction (RT-LAMP and CRISPR/Cas12a) 100% 5 copies/μL ∼45 min No Yes Wang et al. (2021)
smartphone-based CRISPR (1) PCR amplification 100% 1 copy/μL 90 min No No Ma et al. (2022)
(2) CRISPR/Cas fluorescence assay
(3) Colour visualization
RT-PCR/CRISPR-Cas12a-mediated (1) RT-PCR 100% 2 copies/per reaction (∼0.1 copies/μL) >1h No No Liang et al. (2022)
(2) CRISPR-Cas12a-mediated
ECS-CRISPR (1) One-pot reaction (RT-RPA and CRISPR/Cas12) 100% 3 copies/μL 25–30 min No Yes Hu et al. (2022)
OPTIMA-dx (1) One-pot reaction (RT-LAMP and CRISPR/Cas13) 100% 10 copies/μL 45–50 min NA Yes Mahas et al. (2022)
DISCoVER (1)One-pot reaction (rLAMP and Cas13a) 100% 40 copies/μL <50 min NA Yes Chandrasekaran et al. (2022)
OPERATOR (1) One-pot reaction (RCA and CRISPR/Cas fluorescence assay)/LFS 100% 1.625copies per reaction (∼0.081 copies/μL) 30 min Yes Yes This work
Open in a new tab

Notes, NA. Not applicable.

No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part.

A total of 110 clinical samples (S1–S110) were tested using the developed OPERATOR assay. Fig. 4e shows highly significant fluorescence of the positive sample, indicating the presence of SARS-CoV-2. The results show that 74 samples are SARS-CoV-2 positive and 36 samples are negative. The results were completely concordant with those of RT-qPCR analysis (Supplemental Table S4). Among the 74 positive clinical samples, a toal of 53 samples with the Ct values within the range from 30 to 37 are also tested successfully, confirming the good performance of the developed OPERATOR assay for SARS-Cov-2.

3.6. OPERATOR is applicable for accurate on-the-spot visual detection of clinical samples

Since the whole OPERATOR procedure works at the isothermal temperature of 37 °C, we developed a POCT platform for visual on-the-spot detection of SARS-CoV-2 combining rapid virus RNA release and our previously developed LFS (Zhu et al., 2022) with the OPERATOR assay (Fig. 5 a). The POCT included three steps: sample RNA release, OPERATOR reaction, and visual inspection. All three steps can be performed without any dedicated equipment except for a smart thermos cup (HONGPA, Huawei, China). The smart thermos cup can connect to a smartphone through an app to control the reaction temperature and duration. For rapid extraction of virus RNA, we used a commercial RNA quick-release buffer from the icloning Company (Beijing, China). Pharyngeal or nasal swabs need only be dipped into the release buffer for heat treatment at 95 °C for 3 min using a thermos cup. After virus RNA was released into the buffer, 2 μL was transferred to prepare the OPERATOR master mix for target gene amplification. The OPERATOR reaction was performed at 37 °C for 30 min using a thermos cup. After the OPERATOR reaction was finished, the LFS sample pad was dipped into the reaction tube, and results were read visually within 2 min.

Fig. 5.

Fig. 5

Open in a new tab

An on-spot visual OPERATOR platform for SARS-CoV-2 detection. (a) Flowchart of the on-spot visual OPERATOR platform. (b) Specificity evaluation of the on-spot visual OPERATOR platform for SARS-CoV-2 with different respiratory virus RNAs (RM1 to RM7) as the templates. (c) Sensitivity evaluation of the on-the-spot OPERATOR platform with a series of RNA dilutions (162,500, 16,250, 1,625, 162.5, 16.25, 1.625, 0.50 copies/μL) as templates. (d) On-the-spot visual analysis of clinical samples (S1–S10 and S75–S84). P is positive control and N is negative control.

The specificity and sensitivity of the on-the-spot OPERATOR platform were also tested. In specificity tests, a red band at the T line was only observed for OPERATOR products with SARS-Cov-2, but not with the other six respiratory disease viruses (Fig. 5b). In sensitivity tests, red bands at the T line were observed for OPERATOR products at all RNA dilutions except 0.5 copies/μL (Fig. 5c). The LOD was determined as 1.625 copies per reaction, consistent with the fluorescence results.

The developed on-the-spot platform for POCT was also validated using 20 clinical samples (S1–S10 and S75–S84), and a red band at the T line was observed for S1–S10, indicating that all 10 samples were infected with SARS-Cov-2 (Fig. 5d). These results were consistent with those of fluorescence detection (Fig. 4e) and RT-qPCR (Supplemental Table S5), indicating that the visual on-the-spot platform is highly accurate, sensitive, rapid, and suitable for SARS-Cov-2 POCT.

4. Discussion

In this study, we established a novel approach for RNA virus detection, named OPERATOR, and demonstrated its applicability for the identification of SARS-CoV-2. OPERATOR breaks through the concept of traditional RNA detection methods, such as RT-PCR, and RT-LAMP, by integrating RNA-templated DNA ligation, MRCA, and CRISPR/FnCas12a cleavage into a single detection method, achieving superior performance in a reverse transcription-free, ultrasensitive, rapid, highly specific, easy to operate, on-spot, and visual detection method (Table 1).

In OPERATOR, the RNA sequence of interest of the target gene is converted into DNA by RNA-templated DNA ligation instead of conventional reverse transcription, which avoids the reverse transcription step at a specific temperature, enabling a one-pot reaction. Reverse transcription is the most critical step for currently used methods (RT-PCR, RT-LAMP, and RT-RPA), which complicates the procedure and lengthens the duration (Arizti-Sanz et al., 2020; Broughton et al., 2020; Chen et al., 2021; Patchsung et al., 2020; Selvam et al., 2021). OPERATOR innovatively achieves reverse transcription-free RNA testing, which can be extended to detect ssDNA or RNA viruses. In RNA-templated DNA ligation, the RNA sequence of interest is specially converted into DNA with very high specificity. The specificity of RNA-templated DNA ligation is much higher than that of conventional reverse transcription.

OPERATOR isothermally amplifies target sequences using a circular ssDNA as the template in a multiply-primed RCA reaction with a universal random hexamer primer. No special target-specific primers are needed for OPERATOR assays targeting any gene, which makes target DNA amplification independent of a specific target sequence. However, currently used detection methods such as PCR, qPCR, RPA, and LAMP all require primers (Augustine et al., 2020; Wang et al., 2021; Zhang et al., 2022). The high GC content of target genes, complex primer design, and strict reaction conditions all limit the application of rapid nucleic acid detection (Dieffenbach et al., 1993; Yan et al., 2014). Also, our results confirmed that multiply-primed RCA can achieve high yield and amplification efficiency compared with current methods (Supplemental Fig. S3); it can amplify a single molecule to 109 molecules within 10 min (Xu et al., 2021). By contrast, it often takes more than 30 min for amplification by PCR, RPA, and LAMP (Smyrlaki et al., 2020). Thus, using multiply-primed RCA results in ultra-sensitivity for OPERATOR.

OPERATOR creatively identifies the ssDNA of RCA amplicons containing the target gene sequence employing CRISPR/FnCas12a cleavage acting as a reporting sensor, the first example of using a CRISPR/FnCas12a sensor for ssDNA detection. Unlike existing reporting systems for CRISPR/FnCas12a, OPERATOR employs two fluorescent probes (cis- and trans-probes). The cis-probe plays dual roles in dsDNA (cis-probe-hybridized ssDNA) formation and fluorescence signal emission. In OPERATOR, fluorescence is from two sources (cis- and trans-probes), generating double the fluorescence intensity compared with existing reporting systems (Fig. 2d) (Li et al., 2018b). The unique design of dual probes supports different functions in different application scenarios. For example, double fluorescence intensity can be monitored when dual probes are labeled with the same fluorescent dye, which is helpful to improve the sensitivity of detection. When dual probes labeled with different dyes are used, this can effectively improve the specificity of OPERATOR assays and eliminate false positive results because both fluorescence signals should be observed in genuinely positive reactions, while only one fluorescence signal may be observed in false-positive reactions. A dual probe approach can also be extended to develop multiplex OPERATOR assays using multicolor-labeled fluorescent cis-probes. In multiplex OPERATOR design, the cis-probes are specific to various target RNAs and labeled with different fluorescent dyes (FAM, HEX, Cy5, and Cy3, etc), respectively. The corresponding fluorescent signal can be monitored in the reaction with specific target using the equipment with multi-channels. In addition, the CRISPR/FnCas12a cleavage-mediated reporting sensor can be made independent of the PAM motif by introducing a TTT site into the padlock probe, which eliminates the dependence of crRNA design on the target sequence.

Most importantly, we developed a new buffer suitable for three different reactions (padlock probe cyclization, RCA amplification, and CRISPR/FnCas12a cleavage) via step optimization, which makes a one-pot reaction possible. We successfully combined the multistep reactions into a one-pot process, achieving rapid and facile detection of RNA molecules. To our knowledge, few reports have successfully integrated target amplification and CRISPR/FnCas12a cleavage into a one-pot reaction for RNA template analysis due to reaction temperature and enzyme buffer incompatibilities (Liang et al., 2022; Ma et al., 2022; Mahas et al., 2022; Nguyen et al., 2022; Wang, R. et al., 2020). The one-pot reaction greatly shortens the reaction duration, reduces reagent costs, and simplifies the experimental operation. The whole OPERATOR procedure includes three steps and three additional pipetting operations, and it takes more than 3 h for step-wise reactions. By contrast, the OPERATOR reaction takes only 20–40 min with only a single pipetting step. And the reaction duration can be shortened to only 20 min with slightly higher RNA template concentrations (162.5 copies per reaction) (Fig. 4d). The reaction duration is shorter than for most reported POCT methods and RT-qPCR assays (Smyrlaki et al., 2020, van Dongen et al., 2020; Chandrasekaran et al., 2022; Mahas et al., 2022). The one-pot reaction effectively decreases contamination compared with multistep operations and reduces the actual amount and cost of reagents.

The developed OPERATOR assay for SARS-CoV-2 is ultrasensitive, with a LOD of 1.625 copies per reaction (∼0.081 copies/μL), which is more sensitive than recently reported one-pot CRISPR detection (Hu et al., 2022; Wang et al., 2021) and other step-reaction methods (As shown in Table 1). The ultra-sensitivity is mainly attributed to MRCA, and the cis- and trans-cleavage activities of FnCas12a. Multiply-primed RCA is more sensitive than conventional RCA and PCR (Yue et al., 2021). The trans-cleavage activity of FnCas12a can be triggered in the CRISPR/FnCas12a cleavage reaction with a small amount of dsDNA template, which is more sensitive than currently used readout methods for DNA amplicons, such as capillary gel electrophoresis and SYBR Green dyes (Swerdlow and Gesteland, 1990; Wang et al., 2015). In addition, the dual probe design doubles the fluorescence intensity compared with previous CRISPR/Cas9 reporting sensors (Wang, R. et al., 2020). Ultra-sensitivity of OPERATOR is very important for detecting low-abundance samples, especially for the detection of highly contagious viruses at the very early infection stage. The OPERATOR assay for SARS-CoV-2 could identify infection in the early stages, which could effectively reduce the spread of SARS-CoV-2.

OPERATOR benefits from high specificity in principle and practice for analysis of SARS-CoV-2. The specificity of OPERATOR is doubly ensured by RNA-templated DNA ligation and CRISPR/FnCas12a cleavage. In RNA-templated DNA ligation, both ends of the padlock probe must match the sequence of interest for the padlock probe to cyclize and form a circular ssDNA circle. CRISPR/FnCas12a cleavage was already known to be highly specific to the target dsDNA template with guidance from a crRNA, although the resolution of recognition does not reach a single base pair (Zetsche et al., 2015). By combining DNA ligation and CRISPR/FnCas12a cleavage, OPERATOR achieves much higher specificity than currently used methods. In the developed OPERATOR assay targeting the E gene of SARS-CoV-2, only SARS-Cov-2 and its variants were detected, and no fluorescence signal was observed for other respiratory viruses, demonstrating that the developed assay was specific for SARS-CoV-2.

Based on the one-pot reaction of OPERATOR, we established a POCT platform for on-the-spot SARS-CoV-2 detection, in which rapid RNA release and LFS were incorporated into the OPERATOR assay. The whole test procedure was performed using only a simple portable smart thermal cup, and the results were visually read out using LFS. The POCT platform for SARS-CoV-2 was successfully used for clinical swab detection with high accuracy, and the results were completely consistent with those of RT-qPCR analysis. It only took ∼35 min to test all whole clinical samples, which is more time-efficient than the recently reported one-pot in CRISPR test based on RT-LAMP (Chandrasekaran et al., 2022; Mahas et al., 2022). OPERATOR is amenable to on-the-spot detection of SARS-CoV-2, and it could be used for self-testing instead of the currently employed antigen immunoassay strips (Li et al., 2020; Montesinos et al., 2020). Since the post-operation of LFS visualization might bring the risk of contamination, the LFS test is commended to be performed in a relatively independent space. Also, one portable fluorescence reader can be used to inspect the OPERATOR results instead of LFS for POCT.

5. Conclusions

In summary, we successfully developed and verified a novel ultrasensitive and rapid OPERATOR approach, and confirmed its good performance for detecting SARS-CoV-2. OPERATOR achieves reverse transcription-free detection of RNA molecules with high specificity, high sensitivity (1.625 copies per reaction), and high accuracy, and it is rapid. Furthermore, a visual on-the-spot OPERATOR platform was established for the POCT of clinical samples with high accuracy. The developed OPERATOR assay is suitable for identifying SARS-CoV-2, and it has the potential for multiple scenarios, such as family self-testing, environmental trace sample analysis, and rapid screening of large populations. We also believe that the OPERATOR approach can be extended more flexibly to analyze other complex RNA, DNA viruses, microRNAs, and circulating tumor DNA in various research fields by pre-adding the designed PL to the sample's pyrolysis release buffer.

Author contributions

L.-T.Y. and Z.-B.Z. conceived the idea. Z.-B.Z. designed experiments, interpreted the results and performed the experiment, and prepared the original draft. R.L. assisted in performing the experiment of RT-qPCR and clinical sample data assay. Y.-K.G. assisted in the PL design of the E gene. C.W. and Y.-J.W. assisted FnCas12 protein expression and purification. D.-B. Z. assisted in manuscript writing. L.-T.Y. contribute to conceptualization, funding acquisition, resources, supervision, and, writing review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank those who donated the clinical negative samples for this study, and the seven respiratory viruses RMs from the Shanghai Institute of Measurement and Testing Technology (SIMT), Shanghai, China. This work was funded by the Science and technology Innovation 2030 (2022ZD0402001), the Agricultural Research fund of the Science and Technology Commission of Shanghai Municipality, China (21N31900100 and 21N31900200), and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, Shanghai, China.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bios.2023.115179.

Appendix A. Supplementary data

The following is the supplementary data to this article.

Multimedia component 1
mmc1.docx (17MB, docx)

Data availability

Data will be made available on request.

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