Adaptation-coupled CRISPRi identifies antibiotic-sensitizing genes leading to a novel plasmid selection marker
Antibiotic resistance through horizontal gene transfer is a major challenge in microbiology and medicine. We describe a CRISPR-based screening system that couples spacer adaptation with gene repression to identify bacterial genes whose inhibition increases resistance to antibiotics. We used this screening system in E. coli, on seven different antibiotics to select spacers that conferred resistance to them. The resulting spacers targeted a wide array of genes, including those involved in electron transport functions known to be involved in conferring antibiotic resistance, and also new genes not yet known to be involved in antibiotic resistance. We defined this collection of sensitizing genes as the "sensitasome". Some sensitization genes exhibited a clear trade-off , their repression conferred resistance to certain antibiotics while increasing susceptibility to others. Sensitasome-targeting spacers were also effective in Shigella and Salmonella, indicating that the resistance mechanism is conserved across species. Finally, we repurposed a selected spacer as a CRISPR-based antibiotic selection marker using the compact Cas12m effector. This marker is an alternative to traditional resistance genes, reducing horizontal transfer of genes degrading antibiotics, and enabling effective selection at low antibiotic concentrations.