ABSTRACT A big challenge for the control of COVID-19 pandemic is the emergence of variants of concern (VOCs) or variants of interest (VOIs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be more transmissible and/or more virulent and could escape immunity obtained through infection or vaccination. A simple and rapid test for SARS-CoV-2 variants is an unmet need and is of great public health importance. In this study, we designed and analytically validated a CRISPR-Cas12a system for direct detection of SARS-CoV-2 VOCs. We further evaluated the combination of ordinary reverse transcription-PCR (RT-PCR) and CRISPR-Cas12a to improve the detection sensitivity and developed a universal system by introducing a protospacer adjacent motif (PAM) near the target mutation sites through PCR primer design to detect mutations without PAM. Our results indicated that the CRISPR-Cas12a assay could readily detect the signature spike protein mutations (K417N/T, L452R/Q, T478K, E484K/Q, N501Y, and D614G) to distinguish alpha, beta, gamma, delta, kappa, lambda, and epsilon variants of SARS-CoV-2. In addition, the open reading frame 8 (ORF8) mutations (T/C substitution at nt28144 and the corresponding change of amino acid L/S) could differentiate L and S lineages of SARS-CoV-2. The low limit of detection could reach 10 copies/reaction. Our assay successfully distinguished 4 SARS-CoV-2 strains of wild type and alpha (B.1.1.7), beta (B.1.351), and delta (B.1.617.2) variants. By testing 32 SARS-CoV-2-positive clinical samples infected with the wild type ( n = 5) and alpha ( n = 11), beta ( n = 8), and delta variants ( n = 8), the concordance between our assay and sequencing was 100%. The CRISPR-based approach is rapid and robust and can be adapted for screening the emerging mutations and immediately implemented in laboratories already performing nucleic acid amplification tests or in resource-limited settings. IMPORTANCE We described CRISPR-Cas12-based multiplex allele-specific assay for rapid SARS-CoV-2 variant genotyping. The new system has the potential to be quickly developed, continuously updated, and easily implemented for screening of SARS-CoV-2 variants in resource-limited settings. This approach can be adapted for emerging mutations and implemented in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests using existing resources and extracted nucleic acid.
【저자키워드】 SARS-CoV-2, variant screening, emerging SARS-CoV-2 variant, CRISPR-Cas system, 【초록키워드】 public health, coronavirus, vaccination, Mutation, Immunity, B.1.351, COVID-19 pandemic, Sequencing, SARS-CoV-2 variant, Infection, variants of concern, B.1.617.2, severe acute respiratory syndrome Coronavirus, delta variant, RT-PCR, reverse transcription-PCR, variants, Spike protein, Laboratory, CRISPR, amplification, clinical samples, nucleic acid, PCR, Concordance, Rapid test, SARS-CoV-2 variants, L452R, ORF8, B.1.1.7, N501Y, K417N, VOCs, limit of detection, Lineage, D614G, Alpha, variants of interest, E484K, Beta, VOIs, respiratory, multiplex, nucleic acid amplification, nucleic acid amplification tests, resource, genotyping, wild type, Lambda, Delta variants, Amino acid, Combination, Detection sensitivity, Open reading frame, unmet need, protospacer adjacent motif, T478K, acute respiratory syndrome, Frame, acute respiratory syndrome coronavirus, acute respiratory syndrome coronavirus 2, SARS-CoV-2 nucleic acid, primer, SARS-CoV-2 strain, virulent, variants of SARS-CoV-2, approach, robust, IMPROVE, clinical sample, described, detect, indicated, addition, evaluated, PAM, 【제목키워드】 detection, concern, Major,