COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme

Area of Science:

• Molecular Biology
• Virology
• Diagnostic Assays

Background:

• Accurate and rapid identification of SARS-CoV-2 variants is crucial for guiding COVID-19 patient treatment and informing public health strategies.
• Existing methods like whole-genome sequencing can be time-consuming and complex for widespread clinical use.

Purpose of the Study:

• To develop and validate a rapid diagnostic assay for identifying SARS-CoV-2 variants.
• To target specific single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike gene associated with major variants.

Main Methods:

• Development of a DETECTR assay combining loop-mediated isothermal amplification (LAMP) with CRISPR-Cas12 technology.
• Targeting of L452R, E484K/Q/A, and N501Y mutations using the CasDx1 enzyme.
• Validation using 261 clinical samples compared against whole-genome sequencing and RT-PCR.

Main Results:

• The CRISPR-Cas12 based assay accurately identified targeted SNP mutations.
• Achieved 97.3% SNP concordance and 98.9% agreement for SARS-CoV-2 lineage classification in clinical samples.
• Demonstrated that detecting the E484A mutation was sufficient to identify Omicron variants.

Conclusions:

• The CRISPR-based DETECTR assay offers a faster and simpler alternative to sequencing for SARS-CoV-2 variant identification.
• This assay has significant utility for clinical and public health laboratories in managing the COVID-19 pandemic.