4.6. Preparations of SARS-CoV-2 nucleic acid fragments
The specific crRNA used in the study was synthesized and listed in Table S1. The Cas12a crRNA consists of two functional parts: the scaffold region (UAAUUUCUACUAAGUGUAGAU) responsible for binding with the Cas12a protein, and a customized region, which is the reverse complement of the target sequence (~20 nt). The customized region extends from the 3′ end of the scaffold to ensure specificity. The third base in the customized region was designated as the SNP site, and an additional mismatch base was introduced at the fifth position to maximize the difference in cleavage between on-target and off-target sequences. The L452R mutation (24,410 G > A) is unique to the BA.5 Omicron variant and was used as a distinguishing feature between BA.5 and BA.2 variants. The target RNA sequence for detecting the L452R mutation on the spike (S) gene is provided in Table S2. The RNA templates from respiratory pathogens were synthesized through the transcription of the corresponding DNA templates carried on pUC57 plasmids using the HiScribe™ T7 High Yield RNA Synthesis Kit (NEB, USA) (Table S2). After the RNA synthesis, any remaining DNA templates were eliminated by treating the samples with DNase I. Finally, the resulting RNA templates were purified using the Monarch RNA Cleanup Kit (NEB, USA) and subsequently were reverse transcribed into cDNA via the PrimeScript™ RT reagent kit (Takara, Dalian, China).