Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and immune evasion poses a significant global health challenge.
• Accurate and rapid detection of these variants is crucial for effective public health responses, including treatment, isolation, and quarantine measures.

Purpose of the Study:

• To develop a rapid, cost-effective, and instrument-free diagnostic method for the simultaneous detection and differentiation of SARS-CoV-2 variants.
• To validate the performance of the developed assay using clinical samples.

Main Methods:

• Development of a CRISPR-Cas12a-based assay named SAVED (SARS-CoV-2 variants enzymatic detection).
• Utilized chimeric CRISPR RNA (crRNA) and short crRNAs for single nucleotide polymorphism (SNP) genotyping to differentiate variants.
• Targeted key SARS-CoV-2 variants including Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, and Omicron subvariants (BA.1, BA.2, BA.3, BA.4, BA.5).

Main Results:

• The SAVED assay demonstrated 100% specificity across all tested clinical samples.
• Achieved 100% sensitivity for Omicron variant samples with a cycle threshold (Ct) value ≤34.9.
• The assay is inexpensive ($3.23 per reaction), instrument-free, and results are stable for 1 hour, facilitating high-throughput screening and point-of-care use.

Conclusions:

• The SAVED method provides a rapid, sensitive, and specific tool for differentiating SARS-CoV-2 variants.
• Its low cost and instrument-free nature make it suitable for widespread application, including point-of-care testing and high-throughput screening.
• The assay design allows for future modifications to incorporate newly emerging SARS-CoV-2 variants.