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).