Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a
ABSTRACT The continuous and rapid surge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmissibility and evading neutralization is alarming, necessitating expeditious detection of the variants concerned. Here, we report the development of rapid SARS-CoV-2 varia...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-12-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.03260-22 |
| _version_ | 1811195087290368000 |
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| author | Jun Yang Nilakshi Barua Md Nannur Rahman Carmen Li Norman Lo Kai Yan Yeong Tsz Fung Tsang Xiao Yang Yuk-Yam Cheung Alan K. L. Tsang Rickjason C. W. Chan Eddie Chi-Man Leung Paul K. S. Chan Margaret Ip |
| author_facet | Jun Yang Nilakshi Barua Md Nannur Rahman Carmen Li Norman Lo Kai Yan Yeong Tsz Fung Tsang Xiao Yang Yuk-Yam Cheung Alan K. L. Tsang Rickjason C. W. Chan Eddie Chi-Man Leung Paul K. S. Chan Margaret Ip |
| author_sort | Jun Yang |
| collection | DOAJ |
| description | ABSTRACT The continuous and rapid surge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmissibility and evading neutralization is alarming, necessitating expeditious detection of the variants concerned. Here, we report the development of rapid SARS-CoV-2 variants enzymatic detection (SAVED) based on CRISPR-Cas12a targeting of previously crucial variants, including Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, and currently circulating variant of concern (VOC) Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5. SAVED is inexpensive (US$3.23 per reaction) and instrument-free. SAVED results can be read out by fluorescence reader and tube visualization under UV/blue light, and it is stable for 1 h, enabling high-throughput screening and point-of-care testing. We validated SAVED performance on clinical samples with 100% specificity in all samples and 100% sensitivity for the current pandemic Omicron variant samples having a threshold cycle (CT) value of ≤34.9. We utilized chimeric CRISPR RNA (crRNA) and short crRNA (15-nucleotide [nt] to 17-nt spacer) to achieve single nucleotide polymorphism (SNP) genotyping, which is necessary for variant differentiation and is a challenge to accomplish using CRISPR-Cas12a technology. We propose a scheme that can be used for discriminating variants effortlessly and allows for modifications to incorporate newer upcoming variants as the mutation site of these variants may reappear in future variants. IMPORTANCE Rapid differentiation and detection tests that can directly identify SARS-CoV-2 variants must be developed in order to meet the demands of public health or clinical decisions. This will allow for the prompt treatment or isolation of infected people and the implementation of various quarantine measures for those exposed. We report the development of the rapid SARS-CoV-2 variants enzymatic detection (SAVED) method based on CRISPR-Cas12a that targets previously significant variants like Alpha, Beta, Gamma, Delta, Lambda, Mu, and Kappa as well as the VOC Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 that are currently circulating. SAVED uses no sophisticated instruments and is reasonably priced ($3.23 per reaction). As the mutation location of these variations may reoccur in subsequent variants, we offer a system that can be applied for variant discrimination with ease and allows for adjustments to integrate newer incoming variants. |
| first_indexed | 2024-04-12T00:36:41Z |
| format | Article |
| id | doaj.art-b37e27fd3d8e4ac9bfa003377da017ac |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-12T00:36:41Z |
| publishDate | 2022-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-b37e27fd3d8e4ac9bfa003377da017ac2022-12-22T03:55:08ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972022-12-0110610.1128/spectrum.03260-22Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12aJun Yang0Nilakshi Barua1Md Nannur Rahman2Carmen Li3Norman Lo4Kai Yan Yeong5Tsz Fung Tsang6Xiao Yang7Yuk-Yam Cheung8Alan K. L. Tsang9Rickjason C. W. Chan10Eddie Chi-Man Leung11Paul K. S. Chan12Margaret Ip13Department of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, SAR, ChinaDepartment of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, SAR, ChinaDepartment of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, SAR, ChinaDepartment of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, SAR, ChinaDepartment of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, SAR, ChinaDepartment of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, SAR, ChinaDepartment of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, SAR, ChinaDepartment of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, SAR, ChinaMicrobiology Division, Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, Hong Kong, SAR, ChinaMicrobiology Division, Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, Hong Kong, SAR, ChinaMicrobiology Division, Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, Hong Kong, SAR, ChinaDepartment of Microbiology, Prince of Wales Hospital, Hong Kong, SAR, ChinaDepartment of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, SAR, ChinaDepartment of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, SAR, ChinaABSTRACT The continuous and rapid surge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmissibility and evading neutralization is alarming, necessitating expeditious detection of the variants concerned. Here, we report the development of rapid SARS-CoV-2 variants enzymatic detection (SAVED) based on CRISPR-Cas12a targeting of previously crucial variants, including Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, and currently circulating variant of concern (VOC) Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5. SAVED is inexpensive (US$3.23 per reaction) and instrument-free. SAVED results can be read out by fluorescence reader and tube visualization under UV/blue light, and it is stable for 1 h, enabling high-throughput screening and point-of-care testing. We validated SAVED performance on clinical samples with 100% specificity in all samples and 100% sensitivity for the current pandemic Omicron variant samples having a threshold cycle (CT) value of ≤34.9. We utilized chimeric CRISPR RNA (crRNA) and short crRNA (15-nucleotide [nt] to 17-nt spacer) to achieve single nucleotide polymorphism (SNP) genotyping, which is necessary for variant differentiation and is a challenge to accomplish using CRISPR-Cas12a technology. We propose a scheme that can be used for discriminating variants effortlessly and allows for modifications to incorporate newer upcoming variants as the mutation site of these variants may reappear in future variants. IMPORTANCE Rapid differentiation and detection tests that can directly identify SARS-CoV-2 variants must be developed in order to meet the demands of public health or clinical decisions. This will allow for the prompt treatment or isolation of infected people and the implementation of various quarantine measures for those exposed. We report the development of the rapid SARS-CoV-2 variants enzymatic detection (SAVED) method based on CRISPR-Cas12a that targets previously significant variants like Alpha, Beta, Gamma, Delta, Lambda, Mu, and Kappa as well as the VOC Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 that are currently circulating. SAVED uses no sophisticated instruments and is reasonably priced ($3.23 per reaction). As the mutation location of these variations may reoccur in subsequent variants, we offer a system that can be applied for variant discrimination with ease and allows for adjustments to integrate newer incoming variants.https://journals.asm.org/doi/10.1128/spectrum.03260-22CRISPR-Cas12achimeric crRNASARS-CoV-2 variants detectionSAVEDSNP genotypingshort crRNA |
| spellingShingle | Jun Yang Nilakshi Barua Md Nannur Rahman Carmen Li Norman Lo Kai Yan Yeong Tsz Fung Tsang Xiao Yang Yuk-Yam Cheung Alan K. L. Tsang Rickjason C. W. Chan Eddie Chi-Man Leung Paul K. S. Chan Margaret Ip Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a Microbiology Spectrum CRISPR-Cas12a chimeric crRNA SARS-CoV-2 variants detection SAVED SNP genotyping short crRNA |
| title | Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a |
| title_full | Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a |
| title_fullStr | Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a |
| title_full_unstemmed | Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a |
| title_short | Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a |
| title_sort | rapid sars cov 2 variants enzymatic detection saved by crispr cas12a |
| topic | CRISPR-Cas12a chimeric crRNA SARS-CoV-2 variants detection SAVED SNP genotyping short crRNA |
| url | https://journals.asm.org/doi/10.1128/spectrum.03260-22 |
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