Direct visualization of single-cell non-repetitive genes by in situ activation of collateral activity of CRISPR/Cas12a inside cells

The spread of drug-resistance bacteria is a serious issue of environment. Tools allowing to image single-cell genes can provide key information about the spatial pattern and heterogeneity of cell population. Herein, we explored the possibility of in situ activation of collateral trans-cleavage activity of CRISPR/Cas12a inside cells, to achieve a direct detection of single-cell non-repetitive genes. CRISPR/Cas12a allows to recognize target genes without the need for denaturation or digestion process. Particularly, the target gene-activated trans-cleavage by CRISPR/ Cas12a inside cells outputs an amplified signal for the gene recognition, allowing to visualize non-repetitive genes. The signal-to-background ratio for imaging drug-resistance gene, oqxB in the Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) was further improved by combining multiple binding of Cas12a, enabled imaging of drug-resistance S. Typhimurium isolated from poultry farm and in the intestinal tract sec-tions. Single-cell investigation of S. Typhimurium under salt stress indicated that drug-sensitive strain owned a survival advantage over drug-resistance strain at high-content salt environment. This gene imaging methods holds potential for detecting the spread of drug resistance in the environment and serves as a means to inves-tigate the relationship between genotype and phenotype at single-cell level.

Automated summary

This study explores the possibility of directly detecting single-cell non-repetitive genes by using CRISPR/Cas12a to activate collateral trans-cleavage activity inside cells. This method visualizes non-repetitive genes and helps detect the spread of drug resistance in the environment, allowing the investigation of the relationship between genotype and phenotype at the single-cell level.