Chimeric crRNA improves CRISPR–Cas12a specificity in the N501Y mutation detection of Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2

Abstract

Many CRISPR/Cas platforms have been established for the detection of SARS-CoV-2. But the detection platform of the variants of SARS-CoV-2 is scarce because its specificity is very challenging to achieve for those with only one or a few nucleotide(s) differences. Here, we report for the first time that chimeric crRNA could be critical in enhancing the specificity of CRISPR-Cas12a detecting of N501Y, which is shared by Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2 without compromising its sensitivity. This strategy could also be applied to detect other SARS-CoV-2 variants that differ only one or a few nucleotide(s) differences.

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

“…Fluorescence detection is suitable for high-throughput sample sizes. Blue LED light or UV light can also be used to visualize detection tubes, which enables instrument-free detection ( 32 , 37 ). We used tube visualization to read out results instead of lateral flow strips, as the tube visualization apparatus (blue LED light source and transparent orange acrylic sheet) is generally inexpensive and can be recycled ( 32 , 37 ).…”

“…The CRISPR-Cas12a detection mixture was modified from the previous protocols ( 19 , 32 , 42 ). Two different mixtures (low and high concentration) of Cas12a and crRNA were used for crRNA screening and CRISPR-Cas12a detection.…”

“…Fluorescent intensities were monitored every 5 min at 37°C for 1.5 h by Synergy H1 microplate reader (BioTeK) at FAM channel (excitation/bandwidth, 484/12.5; emission/bandwidth, 530/12.5). As for the fluorescent detection in tubes, the 0.2-mL 8-strip tubes were incubated at 37°C before visualizing by ChemiDoc touch imaging system (Bio-Rad) under UV light or blue LED light with a filter, and the detection was performed at 30 min and 1.5 h, respectively ( 32 ).…”

“…Although Cas12a and crRNAs can be engineered to achieve higher specificity, crRNA modification is relatively easier and safer ( 27 – 29 , 31 ). Four crRNA designing strategies were employed to improve the specificity of crRNAs in this study, which include (i) chimeric crRNA (replacing the last 8 nucleotide [nt] RNA of the 24-nt spacer with DNA) ( 28 , 32 ), (ii) short crRNA (15-nt to18-nt spacer) ( 33 ), (iii) 3'DNA7 (7-nt DNA 3′ end extension to crRNA, spacer + 3′ end 7 universal DNA extension [ TATTATT ]) ( 27 ), and (iv) introduction of one or two mismatches in the spacer of crRNA. We used LbCas12a in this study, as LbCas12a has stronger collateral cleavage activity (nonspecific single-stranded DNA [ssDNA] degradation) than AsCas12a and FnCas12a ( 27 , 34 ).…”

“…We aimed to provide an affordable assay that required the fewest instruments with a short turnaround time for simultaneous detection of variants. Initially, we focused on improving the specificity of N501Y detection using chimeric crRNA ( 32 ). We eventually expanded to include mutations in current SARS-CoV-2 variants.…”