| BackgroundCoronavirus disease 2019(COVID-19)pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is a major global public health problem.The virus has been constantly mutating and multiple SARS-CoV-2 variants have been identified.According to their relevant biological properties and public health concern,Alpha,Beta,Gamma,Delta,and Omicron variants have been classified by the World Health Organization(WHO)as variants of concern(VOCs).These variants carry multiple mutations in the Spike protein of SARS-CoV-2 and some of them show enhanced transmissibility and reduced effectiveness to SARS-CoV-2 vaccines,causing different waves of pandemic,which is a new and unsolved threat.Genomic sequencing and phylogenetic analysis ensured our capability to monitor the emergence and spread of SARS-CoV-2 variants.However,their applicability in most resource-limited settings is limited because they are complexed,time-and cost-consuming,and require large equipment and professional technicians.Therefore,it is an urgent and unmet need to develop simple and rapid methods for detecting and tracking SARS-CoV-2 variants.Objectives and significanceThe clustered regularly interspaced short palindromic repeats(CRISPR)and its associated(Cas)system have emerged as next-generation of molecular diagnostics and a powerful tool for the pathogen detection or genotyping by using specific CRISPR RNAs(crRNAs).Our study aimed to develop a recombinase aided amplification(RAA)/CRISPR-Cas12a-mediated assay for highly sensitive and rapid detection of single nucleotide mutation in one pot.Moreover,multiple specific crRNAs were designed and evaluated to detect and identify SARS-CoV-2 major VOCs rapidly and specifically based on the RAA/CRISPR-Cas12a-mediated assay.The significance of our study is to provide a new method and solution for rapid detection and genotyping of SARS-CoV-2 VOCs based on the novel RAA/CRISPRCas12a-mediated assay.In addition,this assay can also be applied for the detection of other pathogens,drug resistance mutations and diagnosis of genetic diseases,which has good application prospects.Methods1.Development of RAA/CRSPR-Cas12a-mediated assay.AsCas12a and LbCas12a proteins were expressed and purified for the development of CRISPRCas12a assay.Then,an artificial prototospacer adjacent motif(PAM)was inserted into the RAA primers in order to amplify any templates without appropriate PAM motif when necessary.Furthermore,the suboptimal APM was designed by inserting one extra additional mutation at the upstream or downstream of the original signature mutation of SARS-CoV-2 variants to enhance the specific detection,which in turn would improve the detection sensitivity and specificity.Eventually,by integrating RAA with CRISPR-Casl2a-based detection in one tube,the assay is operated in ont pot to avoid potential contamination caused by the amplification products.2.Development of RAA/CRISPR-Casl2a assay to detect SARS-CoV-2 VOCs.The spike gene of SARS-CoV-2 was amplified by RAA technology followed by the cleavage of CRISPR-Cas12a assay.5 allele-specific crRNAs(crRNA-417N,crRNA478K,crRNA-484K,crRNA-501N,crRNA-614D)and 2 Omicron-specific crRNAs(crRNA-S-49X and crRNA-50X)targeting the signature mutations in the spike protein of SARS-CoV-2 were identified and selected to detect and distinguish major SARSCoV-2 VOCs including Alpha,Beta,Delta variants,and Omicron sublineages BA.1 and BA.2.3.Performance of RAA/CRISPR-Cas12a assay to detect SARS-CoV-2 VOCs.The low limit of detection(LOD)of RAA/CRIPSR-Cas12a-mediated assay is determined by using 10-fold serial dilutions of the target DNA templates.Moreover,the specificity of the RAA/CRISPR-Cas12a assay was validated with clinical samples infected with 11 common respiratory viruses but not SARS-CoV-2.Finally,we examined 54 SARS-CoV-2 positive clinical samples including 4 samples infected with wild-type strains,16 with Alpha variant,14 with Beta variant,15 with Delta variant and 5 with Omicron variant and compared with Sanger sequencing results.Results1.The RAA/CRISPR-Cas12a-mediated assay has been successfully developed.The whole reaction can be carried out in one tube at 39℃ within 1.5-2 hours and the results can be read out by fluorescence meter or naked eyes.2.Our results show that the RAA/CRISPR-Cas12a-based assay could readily distinguish the signature mutations,i.e.,K417N,T478K,E484K,N501Y and D614G with a low limit of detection(LOD)of 104 copies/μL.When detecting 54 SARS-CoV2 positive clinical samples,the assay showed a concordance of 92.59%(50/54)with Sanger sequencing results.The sensitivity of 88.2%～100%and specificity of 97.5%～100.0%were obtained for the crRNAs analyzed when detecting 5 signature mutations in the spike protein of SARS-CoV-2.3.The two Omicron-specific crRNAs can readily and correctly distinguish Omicron BA.1 and BA.2 sublineages.By using crRNA-S-49X,the RAA/CRISPRCas12a-based assay could specifically detect as low as 10 copies/μL of Omicron BA.1 plasmid DNA without nonspecific cross-reaction.The crRNA-S-50X could readily detect both Omicron BA.1 and BA.2 plasmid with a LOD of 100 copies/μL.Both crRNA-S-49X and crRNA-S-50X could specifically detect Omicron variant in 5 clinical samples infected with BA.1 sublineage and distinguish Omicron variant from 49 clinical samples infected with other SARS-CoV-2 strains but not Omicron.4.Furthermore,no cross reaction was observed for all crRNAs analyzed when detecting clinical samples infected with 11 common respiratory pathogens but not SARS-CoV-2.ConclusionsWe successfully developed a RAA/CRISPR-Cas12a-mediated assay to specifically distinguish major SARS-CoV-2 variants including the prevalent Delta and Omicron sublineage BA.1 and BA.2.All the reactions were conducted in one sealed tube without the need for complex equipment and facilities.The simple and rapid assay could be set up and implemented routinely in resource-limited settings.In the future,this assay can be further simplified and used for high-throughput multiplex screening through combination with sophisticated microfluidic devices. |
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