TY - JOUR
T1 - Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a
AU - Yang, Jun
AU - Barua, Nilakshi
AU - Rahman, Md Nannur
AU - Li, Carmen
AU - Lo, Norman
AU - Yeong, Kai Yan
AU - Tsang, Tsz Fung
AU - Yang, Xiao
AU - Cheung, Yuk Yam
AU - Tsang, Alan K.L.
AU - Chan, Rickjason C.W.
AU - Leung, Eddie Chi Man
AU - Chan, Paul K.S.
AU - Ip, Margaret
N1 - Publisher Copyright:
© 2022 Yang et al.
PY - 2022/11
Y1 - 2022/11
N2 - 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.
AB - 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.
KW - CRISPR-Cas12a
KW - SARS-CoV-2 variants detection
KW - SAVED
KW - SNP genotyping
KW - chimeric crRNA
KW - short crRNA
UR - http://www.scopus.com/inward/record.url?scp=85144636840&partnerID=8YFLogxK
U2 - 10.1128/spectrum.03260-22
DO - 10.1128/spectrum.03260-22
M3 - Article
C2 - 36342324
AN - SCOPUS:85144636840
VL - 10
JO - Microbiology Spectrum
JF - Microbiology Spectrum
IS - 6
ER -
