Detect the SARS-CoV-2 D614G mutation using engineered Cas12a guide RNA

Detection of pathogen-related single-nucleotide variation is indispensable for the disease tracing, but remains as a technical challenge. The D614G mutation in the SARS-CoV-2 genome is known to enhance the viral infectiousness markedly but inconvenient to detect. Here, we report an effective approach for detecting the D614 and G614 variants by using synthetic mismatch integrated crRNA guided Cas12a detection (symRNA-Cas12a). We systemically screened all possible nucleotide substitutions covering -2 to +2 position around mutation in crRNA and got an efficient crRNA, which increased the detection specificity up to 13 folds over ancestral crRNA. With the selected crRNA, the symRNA-Cas12a assay can detect as low as 10 copies of synthetic mutant RNA and has 100% agreement with Sanger sequencing result of SARS-CoV-2 D614G mutants in the clinical samples. Overall, we developed the symRNA-Cas12a to specifically, sensitively and rapidly detect SARS-CoV-2 D614G mutation.