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All issues Volume 29 / No 5 (October 2024) Wuhan Univ. J. Nat. Sci., 29 5 (2024) 453-460 Full HTML
Open Access
Issue
Wuhan Univ. J. Nat. Sci.
Volume 29, Number 5, October 2024
Page(s) 453 - 460
DOI https://doi.org/10.1051/wujns/2024295453
Published online 20 November 2024

© Wuhan University 2024

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

0 Introduction

Since the end of 2019, the coronavirus disease 19 (COVID-19) has seriously affected the lives and health of people around the world. It is one of the major global concerns due to the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronaviruses are large, positive-stranded RNA viruses with genome sizes ranging from 27 to 32 kb[1]. Currently, eight coronaviruses infect humans, comprising three α-coronaviruses (HCOV-229E, HKU-NL63, and CCoV-HuPn-201) and five β-coronaviruses (HCOV-OC43, HCOV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2)[2]. SARS-CoV-2 is a single positive-stranded RNA virus with a genome length of about 30 kb and the coding sequence includes ORF1ab, spike gene (S gene), envelope gene (E gene), membrane gene (M gene), and nucleocapsid gene (N gene)[3,4]. SARS-CoV-2 is highly contagious, especially its mutated strains. Common variants of concern (VOCs) include alpha (B.1.1.7), beta (B.1.351, B.1.351.2, and B.1.351.3), gamma (P.1, P.1.1, P.1.2, P.1.3, P.1.4, P.1.5, P.1.6, and P.1.7), delta (B.1.617.2, AY.1, AY.2, AY.3, and AY.3.1), and omicron (B.1.1.529, BA.1, BA.2, BA.3, BA.4, and BA.5), whereas major Variants of Interest (VOIs) include epsilon (B.1.427/B.1.429), zeta (P.2), eta (B.1.525), theta (P.3), iota (B.1.526), kappa (B.1.617.1), lambda (C.37), and mu (B.162.1)[5]. These mutant strains of viruses spread faster and are more insidious, requiring faster diagnostics to curb the spread of the virus. Early detection and diagnosis are crucial in mitigating the spread of this devastating virus. Accurate diagnosis allows medical staff to deal with patients quickly and governments to take timely measures. Unfortunately, there is always a high risk of a pandemic if delays occur in diagnosis. In order to avoid this situation, a sensitive, specific, rapid, and economical detection method is urgently needed.

Presently, various detection methods for SARS-CoV-2 exist, including gene sequencing, reverse-transcription quantitative PCR (RT-QPCR), enzyme-linked immunosorbent assay (ELISA), and imaging diagnosis[6]. Nucleic acid detection methods, compared with serological methods, are notably accurate and timely, as serological methods rely solely on host-produced antibodies[7]. Due to its sensitivity and efficiency, RT-QPCR is regarded as the gold standard for detecting SARS-CoV-2. Despite its reliability, RT-QPCR still possesses limitations. Personnel technical requirements and experimental equipment limitations make large-scale detection challenging using this method[6,8]. Thus, it remains crucial to develop a novel diagnostic technique that offers efficient, sensitive, accurate, and straightforward tests for rapid diagnosis of SARS-CoV-2 to mitigate disease transmission. Consequently, there is significant interest in developing nucleic acid detection methods utilizing the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins system. Various CRISPR-Cas system technologies, such as CRISPR-Cas12a, CRISPR-Cas12b, and CRISPR-Cas13, have been shown to detect SARS-CoV-2[9-12]. In May 2020, the US food and drug administration (FDA) approved emergency use of a new coronavirus test utilizing CRISPR gene-editing technology[13]. Loop-mediated isothermal amplification (LAMP), conducted at a constant temperature, has recently been employed for point-of-care diagnostics for various pathogens[14].

In this study, we designed a series of LAMP primers and gRNA, and developed the RT-LAMP-CRISPR-Cas12a detection method. The detection system can achieve rapid and accurate detection of SARS-CoV-2 and its mutant strains within one hour. Our work offers the potential for rapid detection of SARS-CoV-2 and its variants.

1 Material and Methods

1.1 Primers and Probes

We used ORF1ab as the target sequence, according to bioinformatics analysis. LAMP primers were designed using the Primer Explorer V5 tool (https://primerexplorer.jp/e/), which were purified with High-Performance Liquid Chromatography. The CRISPR RNA (crRNA) of CRISPR-Cas12a was designed with CHOPCHOP (http://chopchop.cbu.uib.no/). For the fluorescence readout of Lachnospiraceae bacterium Cas12a (LbCas12a) mediated detection, we also designed an ssDNA probe that could be cut, leading to fluorescence upon specific activation of LbCas12. All primers, ssDNA probe, and gRNA were synthesized by Huayu Gene. All sequence data were uploaded to NCBI and GISAID.

1.2 RT-LAMP Assay

RT-LAMP reactions are performed on ice. The detailed operating process is as follows: Each 25 μL reaction contained 8 U/μL of Bst4.0 DNA/RNA Polymerase (HaiGene), 10× Isothermo Buffer (HaiGene), 8 mmol/L Mg2+ (HaiGene), 10 mmol/L dNTP Mix (TaKaRa), 1 μg of sample, 12 μL of primer mix (F3/B3 0.8 μmol/L each, FIP/BIP 1.6 μmol/L each), 2 μL of DEPC water (DNase/RNase free water) (Biosharp). The multiplexed reaction was conducted at 63 ℃ for 40 min. Negative controls were included on each experiment. Positive samples would exhibit color changes.

1.3 CRISPR-Cas12a Assay

CRISPR-Cas12a assay was composed by 0.2 μmol/L Engen LbCas12a (Cpf1) (New England Biolabs, USA), 1×NEBuffer2.1 Reaction Buffer (New England Biolabs, USA), 0.6 μmol/L gRNA, 1 μmol/L ssDNA, 2 μL sample. After the LbCas12a-gRNA complex was incubated at 37 ℃ for 5 min, ssDNA was added, followed by the addition 2 μL of LAMP product to achieve a final volume of 20 μL. The reaction was carried out at 37 ℃ for 30 min. The fluorescence value was quantified by BiosystemsTM QuantStudioTM 3 & 5.

1.4 Viral RNA of SARS-CoV-2

The SARS-CoV-2 mRNA samples used in this experiment were donated by Professor Zhou Li from Center of Animal Experiment /Animal Biosafety level-III Laboratory of Wuhan University.

2 Results

2.1 Establishment of the RT-LAMP-CRISPR-Cas12a Platform

The process of virus amplification and sensing consisted of two steps. We used RNA extracted from nasopharyngeal or oropharyngeal swabs in a universal transport medium (UTM) as samples. Firstly, we added virus RNA in the Eppendorf tube. This tube contained the reagents needed for the RT-LAMP reaction and set the reaction at 63 ℃ in the temperature control unit for 40 min. Next, another sequence-specific recognition process was achieved through a gRNA that recruited a Cas12a protein and activated its DNA cutting activity. Then the ssDNA linked to the fluorescent groups and quenched groups is cleaved, and the fluoresce can be detected (Fig. 1(a)).

thumbnail Fig. 1 Process of RT-LAMP-CRISPR-Cas12a detection platform

(a) Schematic of RT-LAMP-CRISPR-Cas12a detection workflow. (b) The genome map shows the SARS-CoV-2 architecture. The target site and gRNA sequence on the SARS-CoV-2 S gene is highlighted. (c) The sequences and location of RT-LAMP primers and gRNA

We designed a series of RT-LAMP primers and applied bioinformatics to analyze RT-LAMP primers. In the following experiments, we selected a set of 4 RT-LAMP primers for SARS-CoV-2 target amplification and crRNA targeting ORF1ab gene for CRISPR-Cas12a detection (Fig. 1(b), 1(c)).

2.2 Detection of SARS-CoV-2

We used MEGA7 software to perform sequence alignment of the selected primers and explore their specificity in different coronavirus and SARS-CoV-2 mutant strains. The comparison results showed that among the seven coronaviruses infecting humans, the primers we designed only existed in a novel coronavirus with high specificity (Fig. 2(a)). Moreover, analysis in VOCs evolved from novel coronavirus original strains, which showed that the primers and target sequences were highly conserved in the variant strains (Fig. 2(b)). These results demonstrated that the primers we designed could probably detect novel coronavirus variants of VOCs.

thumbnail Fig. 2 Establishment of the RT-LAMP-CRISPR-Cas12a platform to detect SARS-CoV-2

(a) Analysis of primers specificity in seven human-infecting coronaviruses. (b) Analysis of primers conservation in VOC variant strains of SARS-CoV-2.Note: Wuhan-Hu-1: This strain is the world's first whole genome sequence of the new coronavirus shared by China with the world

2.3 Validation of the RT-LAMP-CRISPR-Cas12a Platform

Then we carried out a LAMP experiment to verify these primers. According to literature[15], the optimal reaction temperature for LAMP is 60-65 ℃. We set up different LAMP reaction systems accordingly and finally determined the reaction system at 63 ℃ for a 30 min constant temperature reaction. We used this system for these primers' specificity detection, and the results showed no change in SARS-CoV-2 negative mRNA. There were apparent bands in the SARS-CoV-2 positive mRNA sample group, indicating that our primers have high specificity (Fig. 3(a)).

thumbnail Fig. 3 RT-LAMP-CRISPR-Cas12a platform detection

(a) LAMP experiments demonstrated primer specificity. NC: control, PC: SARS-CoV-2 positive mRNA was added. (b) Optimization of the ssDNA probe concentration. A: Blank control; B: Experimental group 4. Add NEBuffer2.1, Cas12a, gRNA, 10 μL of ssDNA, 10 μL of LAMP product; C: Experimental group 3. Add NEBuffer2.1, gRNA, 5 μL of ssDNA, 10 μL of LAMP product; D: Negative control group. Add NEBuffer2.1, Cas12a, gRNA, ssDNA, and LAMP products of SARS-CoV-2 negative samples; E: Experimental group 1. Add NEBuffer2.1, Cas12a, gRNA, 10 μL of LAMP product; F: Experimental group 2. Add NEBuffer2. 1, Cas12a, gRNA, 1 μL of ssDNA,10 μL of LAMP product; G: Experimental group 5. Add NEBuffer2.1, Cas12a, gRNA, 15 μL of ssDNA, 10 μL of LAMP product; H:Experimental group 6. Add NEBuffer2.1, Cas12a, 10 μL of ssDNA, 10 μL of LAMP product. (c) Quantitative verification of RT-LAMP-CRISPR-Cas12a system. NC: Negative control group. Add NEBuffer2.1, Cas12a, gRNA, ssDNA, and LAMP products of SARS-CoV-2 negative samples; E1: Experimental group 1. Add NEBuffer2.1, Cas12a, gRNA, ssDNA; E2: Experimental group 2. Add NEBuffer2.1, Cas12a, gRNA, 10 μL of LAMP product; E3: Experimental group 3. Add NEBuffer2.1, gRNA, ssDNA, 10 μL of LAMP product; E4: Experimental group 4. Add NEBuffer2.1, Cas12a, gRNA, ssDNA, 10 μL of LAMP product. ****P<0.000 1. (d) Observing the effects of varying ssDNA concentrations under UV irradiation. NC: Negative control group. Add NEBuffer2.1, Cas12a, gRNA, ssDNA, and LAMP products of SARS-CoV-2 negative samples; E1: Experimental group 1. Add NEBuffer2.1, Cas12a, gRNA, 10 μL of LAMP product; E2: Experimental group 2. Add NEBuffer2. 1, Cas12a, gRNA, 1 μL of ssDNA,10 μL of LAMP product; E3: Experimental group 3. Add NEBuffer2.1, gRNA, 5 μL of ssDNA, 10 μL of LAMP product; E4: Experimental group 4. Add NEBuffer2.1, Cas12a, gRNA, 10 μL of ssDNA, 10 μL of LAMP product; E5: Experimental group 5. Add NEBuffer2.1, Cas12a, gRNA, 15 μL of ssDNA, 10 μL of LAMP product

The final RT-LAMP-CRISPR-Cas12a platform reaction system is as follows: 2 μg of sample was amplified by RT-LAMP, reacted at 63 ℃ for 30 min, taking 15 μL of RT-LAMP reaction mixture (fluorescent dye-free) to add Cas12a containing 0.2 μmol/L EnGenLba Cas12a, 1×NEB buffer 2.1, 0.6 μmol/L gRNA, 1 μmol/L ssDNA, 8 U of RNase inhibitor in 25 μL of Cas12a cleavage system. The Cas12a cleavage reaction was performed at 37 ℃ for 5 min, and the change in fluorescence value was detected (Fig. 3(c)). After that, we optimized the reaction system by changing the ssDNA concentration. These results showed that with the increase of the ssDNA probe concentration, the fluorescence value increased (Fig. 3(b)). When the concentration increased to 5 μmol/L, the color change could be observed with the naked eye under UV irradiation (Fig. 3(d)).

3 Discussion

Numerous reports have demonstrated the applicability of CRISPR technology as a testing platform for SARS-CoV-2 infection[12,16,17], primarily attributed to the cis and trans cleavage activities of Cas12 and Cas13 effectors[18-22]. However, its sensitivity is relatively low. LAMP isothermal nucleic acid amplification technology enables ultra-sensitive and rapid detection of target sequences, albeit with barely satisfactory specificity. The combination of these technologies effectively addresses their respective limitations. Previous studies have demonstrated the ability of the RT-LAMP-CRISPR-Cas platform to detect the original strain of SARS-CoV-2. Some studies have optimized the CRISPR detection system, such as Tong et al constructed the AapCas12b protein mutant and found that by reducing the interaction force of the Cas12b protein with the substrate, its cis-cleavage ability can be significantly reduced. In mutant-mediated one-step detection, the detection sensitivity is increased by 10-10 000 fold, and the LAMP technology using only a single enzyme is less costly and has greater clinical translational potential[23]. However, its efficacy against all mutant strains remains unknown (Table 1). This paper presents the design of LAMP primers and gRNA for developing the RT-LAMP-CRISPR-Cas12a assay, enabling rapid and accurate detection of SARS-CoV-2 and its mutant strains within one hour. Our aim is rapid detection of SARS-CoV-2 and its five variants, including SARS-CoV-2 (NC_045512), Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). Our target sequence has been shown to be present in six viral variants (Fig. 2) and it is also interesting to exist in a new variant of Omicronx strain BA.1 (data unshown), suggesting that our detection platform may be suitable for future novel SARS-CoV-2 variants, and this work lays the foundation for the possible emergence of new virus pandemics in the future. Researchers have mitigated off-target effects by enhancing the specificity of guide RNA and implementing multi-target screening[24]. Nucleic acid extraction is currently required for CRISPR diagnostics, and researchers should explore more convenient methods that do not require isolation of DNA or RNA in order to facilitate CRISPR-based detection. Portable cartridges and lyophilized reagents can be further developed for convenient testing outside clinical diagnostic laboratories in the future.

Table 1

Comparison of different detection methods based on CRISPR-Cas system

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All Tables

Table 1

Comparison of different detection methods based on CRISPR-Cas system

In the text

All Figures

thumbnail Fig. 1 Process of RT-LAMP-CRISPR-Cas12a detection platform

(a) Schematic of RT-LAMP-CRISPR-Cas12a detection workflow. (b) The genome map shows the SARS-CoV-2 architecture. The target site and gRNA sequence on the SARS-CoV-2 S gene is highlighted. (c) The sequences and location of RT-LAMP primers and gRNA
In the text
thumbnail Fig. 2 Establishment of the RT-LAMP-CRISPR-Cas12a platform to detect SARS-CoV-2

(a) Analysis of primers specificity in seven human-infecting coronaviruses. (b) Analysis of primers conservation in VOC variant strains of SARS-CoV-2.Note: Wuhan-Hu-1: This strain is the world's first whole genome sequence of the new coronavirus shared by China with the world
In the text
thumbnail Fig. 3 RT-LAMP-CRISPR-Cas12a platform detection

(a) LAMP experiments demonstrated primer specificity. NC: control, PC: SARS-CoV-2 positive mRNA was added. (b) Optimization of the ssDNA probe concentration. A: Blank control; B: Experimental group 4. Add NEBuffer2.1, Cas12a, gRNA, 10 μL of ssDNA, 10 μL of LAMP product; C: Experimental group 3. Add NEBuffer2.1, gRNA, 5 μL of ssDNA, 10 μL of LAMP product; D: Negative control group. Add NEBuffer2.1, Cas12a, gRNA, ssDNA, and LAMP products of SARS-CoV-2 negative samples; E: Experimental group 1. Add NEBuffer2.1, Cas12a, gRNA, 10 μL of LAMP product; F: Experimental group 2. Add NEBuffer2. 1, Cas12a, gRNA, 1 μL of ssDNA,10 μL of LAMP product; G: Experimental group 5. Add NEBuffer2.1, Cas12a, gRNA, 15 μL of ssDNA, 10 μL of LAMP product; H:Experimental group 6. Add NEBuffer2.1, Cas12a, 10 μL of ssDNA, 10 μL of LAMP product. (c) Quantitative verification of RT-LAMP-CRISPR-Cas12a system. NC: Negative control group. Add NEBuffer2.1, Cas12a, gRNA, ssDNA, and LAMP products of SARS-CoV-2 negative samples; E1: Experimental group 1. Add NEBuffer2.1, Cas12a, gRNA, ssDNA; E2: Experimental group 2. Add NEBuffer2.1, Cas12a, gRNA, 10 μL of LAMP product; E3: Experimental group 3. Add NEBuffer2.1, gRNA, ssDNA, 10 μL of LAMP product; E4: Experimental group 4. Add NEBuffer2.1, Cas12a, gRNA, ssDNA, 10 μL of LAMP product. ****P<0.000 1. (d) Observing the effects of varying ssDNA concentrations under UV irradiation. NC: Negative control group. Add NEBuffer2.1, Cas12a, gRNA, ssDNA, and LAMP products of SARS-CoV-2 negative samples; E1: Experimental group 1. Add NEBuffer2.1, Cas12a, gRNA, 10 μL of LAMP product; E2: Experimental group 2. Add NEBuffer2. 1, Cas12a, gRNA, 1 μL of ssDNA,10 μL of LAMP product; E3: Experimental group 3. Add NEBuffer2.1, gRNA, 5 μL of ssDNA, 10 μL of LAMP product; E4: Experimental group 4. Add NEBuffer2.1, Cas12a, gRNA, 10 μL of ssDNA, 10 μL of LAMP product; E5: Experimental group 5. Add NEBuffer2.1, Cas12a, gRNA, 15 μL of ssDNA, 10 μL of LAMP product
In the text

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