Figure 1

SARS-CoV-2 Variant of Concern identification by VarLOCK assay. (A) illustration of the VarLOCK assay. The region with wildtype (green) or mutant (pink) sequence of SARS-CoV-2 RNA is amplified by either RPA, LAMP or PCR. Amplified DNA fragments are subjected to SHERLOCK assay with a pair of gRNAs matching the wildtype sequence (wt gRNA, green) or the mutant sequence (VOC gRNA, pink). We aimed to find conditions in which only a perfect match between the gRNA and the amplified DNA can trigger a collateral nuclease activation of the Cas12b protein to cleave a quenched fluorescent reporter. This allows nuclease activity to be monitored by the increase in fluorescence. The ratiometric response of an unknown SARS-CoV-2 variant sample indicates the presence, or absence, of the targeted mutation and thus enables identification of the VOC. (B) Schematic of the genome of SARS-CoV-2 (left) with blow-up of the Spike protein encoding region showing the location of RBD, S1 and S2. Nineteen mutation sites were chosen for VarLOCK assay for which gRNA pairs were designed and optimised. Nucleotide sequences encoding the relevant amino acids are shown in the table and the nucleotide substitutions/deletions are marked in red. The association of the mutations with various variants of concern is indicated in the table, creating a barcode-like identification matrix. (C) Application of this approach to saliva samples successfully identifies original Wuhan, Alpha, Beta, Delta and Omicron variants with results confirmed by sequencing.