Direct visualization of living bacterial genotypes using CRISPR/Cas12a-circular reporter nanoprobes

Bacterial genotyping is important for understanding the complex microbiota. Although fluorescence in situ hybridization (FISH) has enabled bacterial community identification with high spatial resolution, its unavoidable cell fixation steps and signal generation by multi-probe stacking greatly limit its application in living bacterial genotyping. Here, we designed polyethyleneimine-encapsulated CRISPR/Cas12a-circular reporter nanoprobes (CasCLR) for rapid and sensitive visualization of gene information in living bacteria. We found that, nanoprobe-based sequential delivery of Cas12a/crRNA and circular reporter into bacteria allowed single genomic loci to initiate trans-cleavage activity of Cas12a, thereby cleaving CLR to generate amplified fluorescent signals for imaging of target gene. Using CasCLR, we can sensitively analyze the percentage of target bacteria in co-culture experiments and directly detect pathogenic bacteria in uncultured mouse gut microbe. In addition, CasCLR has the ability to sensitively analyze specific genotype of microbial communities in vivo. This nanobiotechnology-based bacterial gene analysis is expected to advance understanding of in vivo bacterial cytogenetic information.