Detection of the SARS‐CoV‐2 D614G mutation using engineered Cas12a guide RNA

Abstract

Detection of pathogens with single‐nucleotide variations is indispensable for the disease tracing, but remains technically challenging. The D614G mutation in the SARS‐CoV‐2 spike protein is known to markedly enhance viral infectivity but is difficult to detect. Here, we report an effective approach called “synthetic mismatch integrated crRNA guided Cas12a detection” (symRNA‐Cas12a) to detect the D614 and G614 variants effectively. Using this method, we systemically screened a pool of crRNAs that contain all the possible nucleotide substitutions covering the ‐2 to +2 positions around the mutation and identify one crRNA that can efficiently increase the detection specificity by 13‐fold over the ancestral crRNA. With this selected crRNA, the symRNA‐Cas12a assay can detect as low as 10 copies of synthetic mutant RNA and the results are confirmed to be accurate by Sanger sequencing. Overall, we have developed the symRNA‐Cas12a method to specifically, sensitively and rapidly detect the SARS‐CoV‐2 D614G mutation.

Current diagnostic approaches to detect two important betacoronaviruses: Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

“…Specificity: Unclear [309] Almost all variants 69del, 70del, Y144del, N501Y and A570D Compared to WGS, RT-PCR and sanger sequencing require shorter time and lower cost. LOD unclear [310] SNP genotyping Almost all variants Unreported >62% to be able to distinguish two variants of different genotypes [311] RT-LAMP P.1 (Gamma), P.2 (Zeta) N501Y, E484K/Q, K417N/T Sensitivity: 97%, Specificity: 100% (based on N/E targets) [312] B.1.1.7 (Alpha) 69del, 70del LOD: 39-10,000 RNA copies/reaction [313] CRISRP/Cas-based technique B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) N501Y LOD, sensitivity and specificity unclear [346] Almost all variants have D614G D614G LOD: 10 RNA copies/reaction [314] Other variants E174R/S542R/K548R, S254F LOD: 50-1000 RNA copies/reaction, specificity: 100% [315] Almost all variants have D614G D614G LOD: 82 RNA copies/reaction, % specificity unclear [155] Antigen test B.1.1.7 (Alpha), B.1.351 (Beta), Unclear LOD: 1.7×10 5 – 6.6×10 7 RNA copies/mL [316] Sensitivity and specificity: Unclear Antigen and antibody test B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) Unclear Detection rate of antigen/antibody tests to detect these three variants were reported to be >90% [317] …”

“…The LOD of this test approach was reported to be ranging from 39 to 10,000 RNA copies in a reaction [313] , and the test sensitivity and specificity were shown to be around 97, and 100%, respectively [312] . On the other side, a molecular diagnostic approach that is based on the CRISPR/Cas-based detection technique was also being employed in identifying different SARS-CoV-2 variants and the LOD of this test was reported to vary from 10 to 1000 RNA copies in a reaction [155] , [314] , [315] . The specific binding and interaction between the Cas enzyme and the RNA strand allow the specific recognition of a unique virus genome mutation [155] .…”

COVID-19 one year later: a retrospect of CRISPR-Cas system in combating COVID-19

“…Wang et al proposed a method based on a CRISPR-Cas13 amplification principle to profile mutated variants of SARS-CoV-2 54 . At the same time, Meng et al used engineered Cas12a gRNA to detect the SARS-CoV-2 D614G mutation 55 .…”

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform

“…When combined with a nonspecific ssDNA reporter, Cas12a has been used to detect nucleic acids reliably ( Bai et al, 2019 ; Ding et al, 2021 ; Gong et al 2021a , 2021b ; Ma et al, 2021 ; Wang et al, 2021b ). By introducing an additive mismatch in crRNA, the CRISPR/Cas12 system can even be used for single-nucleotide polymorphism (SNP) genotyping with single-base specificity ( Chen et al, 2021 ; Huang et al, 2021 ; Lee et al, 2020 ; Lee Yu et al, 2021 ; Meng et al, 2021b ). The identification of a single base makes Cas12 an ideal approach for mutation detection.…”