Detection of Major SARS-CoV-2 Variants of Concern in Clinical Samples via CRISPR-Cas12a-Mediated Mutation-Specific Assay

Abstract

Objectives: Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a great threat and burden to global public health. Here, we evaluated a clustered regularly interspaced short palindromic repeat-associated enzyme 12a (CRISPR-Cas12a)-based method for detecting major SARS-CoV-2 variants of concern (VOCs) in SARS-CoV-2 positive clinical samples. Methods: Allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of SARS-CoV-2 are designed. A total of 59 SARS-CoV-2 positive oropharyngeal swab specimens were used to evaluate the performance of the CRISPR-Cas12a-mediated assay to identify major SARS-CoV-2 VOCs. Results: Compared with Sanger sequencing, the eight allele-specific crRNAs analyzed can specifically identify the corresponding mutations with a positive predictive value of 83.3−100% and a negative predictive value of 85.7−100%. Our CRISPR-Cas12a-mediated assay distinguished wild-type and four major VOCs (Alpha, Beta, Delta, and Omicron) of SARS-CoV-2 with a sensitivity of 93.8−100.0% and a specificity of 100.0%. The two methods showed a concordance of 98.3% (58/59) with a κ value of 0.956−1.000, while seven (11.9%) samples were found to be positive for extra mutations by the CRISPR-based assay. Furthermore, neither virus titers nor the sequences adjacent to the signature mutations were associated with the variation of fluorescence intensity detected or the false-positive reaction observed when testing clinical samples. In addition, there was no cross-reaction observed when detecting 33 SARS-CoV-2 negative clinical samples infected with common respiratory pathogens. Conclusions: The CRISPR-Cas12a-based genotyping assay is highly sensitive and specific when detecting both the SARS-CoV-2 wild-type strain and major VOCs. It is a simple and rapid assay that can monitor and track the circulating SARS-CoV-2 variants and the dynamics of the coronavirus disease 2019 (COVID-19) pandemic and can be easily implemented in resource-limited settings.

CRISPR-Cas12a-Empowered Electrochemical Biosensor for Rapid and Ultrasensitive Detection of SARS-CoV-2 Delta Variant

“…These results demonstrated that the established detection platform was capable of testing the SARS-COV-2 Delta variant with high specificity. Meanwhile, due to the programmability of crRNA, Cas-based biosensors allow the development of a general biosensing platform for any other emerging SARS-CoV-2 variants by easily changing the crRNA guide region sequence in theoretical [54,55]. Moreover, the long-time stability of in vitro diagnostic devices is critical for practical applications.Hence, the stability and reproducibility of the AuNPs-enforced E-CRISPR platform have been characterized.…”

The Potential of Nanobodies for COVID-19 Diagnostics and Therapeutics

“…Thereafter, Liang et al investigated the use of the CRISPR-Cas12a assay for detection of the omicron variant [ 32 ], and the results indicated that one crRNA containing 3–4 mutations was able to identify and diagnose the variant [ 32 ]. Finally, the CRISPR-Cas12a assay was shown to detect major variants of concern (Alpha, Beta, Delta, and Omicron) in clinical samples [ 33 ]. Wang et al described the detection method, light-up CRISPR-Cas13 transcription amplification, which can target and identify SARS-CoV-2 as well as mutated variants [ 34 ].…”

CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern

“…The trans -cleavage activity of Cas12a facilitates the cleavage of reporter probes and generates amplified signals. The sensitivity of these assays is further improved by integrating CRISPR-Cas12a with nucleic acid amplification techniques, including PCR [ [134] , [135] , [136] , [137] ], recombinant polymerase amplification (RPA) [ 27 , 107 , 138 ], and loop-mediated isothermal amplification (LAMP) [ 34 , 139 ]. The viral RNA is reverse transcribed, the complementary DNA is amplified, and the amplicons are subsequently detected ( Fig.…”

“…Single-nucleotide mismatches within the first six bases proximal to PAM significantly impacted the trans -cleavage activity of Cas12a [ 27 ]. On the basis of this finding, Liang et al [ 136 ] developed a CRISPR-Cas12a-mediated mutation-specific assay and detected six mutations in a total of 59 clinical samples involving the wild-type and four major variants. In their design of crRNA, the authors considered targeting six mutations: E484K, K417 N, L452R, T478K, N501Y, and D614G, for the differentiation of the four variants.…”