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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) such as the Alpha, Beta, Gamma, Delta, and Omicron variants, have been reported to affect viral transmissibility, diagnostics, disease severity, azithromycin summary of product characteristics and vaccine efficacy.

Fast and accurate detection of SARS-CoV-2 variants is crucial for gathering information on VOC-led outbreaks and potential lines of transmission. An effective, rapid, and low-cost diagnostic tool that can accurately detect SARS-CoV-2 variants is therefore important in the prevention and control of infection.

Study: Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform. Image Credit: Mongkolchon Akesin / Shutterstock.com

About the study

In a recent study published in the journal Biosensors and Bioelectronics, scientists developed an improved biosensor with Cas12a-based clustered regularly interspaced short palindromic repeats (CRISPR) on-site rapid detection system (CORDS) and discuss its ability to detect SARS-CoV-2 VOCs. This bio-sensing system was developed by combining Cas12a-based reverse transcription-polymerase chain reaction (RT-PCR) with CORDS.

Since CRISPR/Cas12a is capable of differentiating single nucleotide polymorphisms (SNPs) with the single-base resolution, scientists used key mutations of SARS-CoV-2 VOCs including 69/70 deletion, N501Y, and D614G to verify the ability of CRISPR to identify SARS-CoV-2 mutations.

The reference genome sequence of SARS-CoV-2 was downloaded from the National Center for Biotechnology Information, whereas SARS-CoV-2 variant mutation information was obtained from the Global Initiative on Sharing All Influenza Data (GISAID).

Efficacy of CRISPR/Cas12a to identify SARS-CoV-2 mutations

The type-specific CRISPR RNA (crRNA) sequences were designed to target wild-type (WT) and mutant-type (MT) sequences for each mutation of SARS-CoV-2. A skillful mismatch was added in 501 and 641 for easy differentiation of WT from MT, respectively. An in vitro cleavage assay was performed to verify the performance of the CRISPR/Cas12a system to identify SARS-CoV-2 mutations.

The results of the assay confirmed that 501-crRNA-W, 501-crRNA-M, 614-crRNA-W, 614-crRNA-M, 69/70-crRNA-W, and 69/70-crRNA-M specifically and efficiently cleave at their respective target substrates N501-WT, Y501-MT, D614-WT, G614-MT, 69/70-WT, and 69/70-MT, respectively.

CORDS displayed high specificity to detect DNAs target of SARS-CoV-2 mutations

To determine the specificity of Cas12a, the researchers incorporated fluorescence probes, 12-nt single-stranded DNA (ssDNAs) labeled with 5′-FAM and 3′-BHQ, and a fluorescence reporter system to detect Cas12a activity. They found increased fluorescence activity for substrates Y501-MT, N501-WT, 69/70-WT, and 69/70-MT while using 501-crRNA-M, 501-crRNA-W, 69/70-crRNA-W, and 69/70-crRNA-M, respectively.

Surprisingly, the G614-MT substrate only activated 614-crRNA-M, while D614-MT was slightly activated by 614-crRNA-W. Although a significant difference in fluorescence intensity was observed between 614-crRNA-W and 614-crRNA-M, no significant impact was observed in D614G mutation identification.

The kinetic data displayed that CORDS can accurately differentiate MT variants from WT within 30 minutes. This indicated that CORDS is a rapid and accurate platform for detecting SARS-CoV-2 mutations.

CORD displayed high sensitivity to detect DNAs target of SARS-CoV-2 mutations

To further determine the sensitivity of CORDS, scientists used PCR amplification. The limit of detection (LOD) was 10^-16 M for the Y501-MT and N501-WT amplified substrates and that for the 69/70 deletion using TTC as the protospacer adjacent motif (PAM) was 10^-17 M. This indicated that the CORDS fluorescence reporting system can rapidly detect mutations from SARS-CoV-2 variants with high sensitivity and specificity.

The authors combined the lateral flow strips as a substitute for the fluorescence reporting system. In order to determine its sensitivity, they used 5′-digoxin and 3′-biotin-labelled 14-nt ssDNA and lateral flow strips to detect Cas12a activity.

A LOD of 10^-15 M was observed for Y501-MT, N501-WT, and 69/70-MT substrates. This indicated the high sensitivity of the CORDS paper strips system to rapidly detect SARS-CoV-2 variant mutations, thus eliminating the need for a signal detection instrument.

RT-CORDS is highly accurate and sensitive to detect SARS-CoV-2 mutations

The RT-CORDS assay comprises an RT-PCR instead of a PCR reaction and revealed increased sensitivity of 10^-17 M for the N501Y mutation, which changed to 10^-15 M with the use of lateral flow strips. The sensitivity of the RT-CORDS fluorescence assay for the 69/70 deletion was 10^-17 M and had a LOD of  10^-16 M with the RT-CORDS lateral flow strip. The sensitivity for the D614G detection was 10^-17 M and the RT-CORDS lateral flow strip was 10⁻¹⁵ M.

The kinetic analyses for the identification of N501Y and 69/70 deletion displayed a shorter reaction time for mutation tracking and can be identified in 5-10 minutes. However, 40 min was suggested for D614G detection.

Overall, the current study showed that RT-CORDS is an accurate, sensitive, rapid, and low-cost diagnosis tool for the identification of SARS-CoV-2 single-base mutations N501Y, D614G, and deletion 69/70 using CRISPR/Cas12a, thereby making it an ideal choice for high throughput SARS-CoV-2 variant screening.

Written by

Susha Cheriyedath

Susha has a Bachelor of Science (B.Sc.) degree in Chemistry and Master of Science (M.Sc) degree in Biochemistry from the University of Calicut, India. She always had a keen interest in medical and health science. As part of her masters degree, she specialized in Biochemistry, with an emphasis on Microbiology, Physiology, Biotechnology, and Nutrition. In her spare time, she loves to cook up a storm in the kitchen with her super-messy baking experiments.