Single-Cas, Single-Reporter, and Time-Resolved CRISPR-Cas by Stoichiometry Coding of Multiplex crRNAs in a Single Tube for <i>Brucella</i> Species and ABO Genotype Identification

Genotyping is significant in identifying pathogen gene types and disease-related allele genes. Multiplex nucleic acid detection is convenient and useful for genotyping. Though CRISPR-Cas has great advantages in nucleic acid detection, multiplex detection strategies remain to be developed. Here, we propose a novel CRISPR-Cas strategy characterized by using stoichiometry coding of multiplex crRNAs. Different targets can be recognized by the corresponding crRNA with a certain amount, leading to distinguishable fluorescence difference. We employed this strategy to achieve <i>Brucella</i> species identification and human cell ABO blood genotyping. The detection can be conveniently visualized in a time-resolved way using a single tube, single Cas, and single reporter. The strategy can be employed in a PCR-coupled, RAA-coupled, or amplification-free way. The multiplex crRNA-coding CRISPR-Cas has potential application in differentiating gene types in various fields.