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 that sensitize bacteria to antibiotics. We used this screening system in Escherichia coli, on seven different antibiotics to select spacers that conferred resistance to them. The screen identified both known and previously unrecognized genes that modulate antibiotic susceptibility. We defined this collection of genes that sensitize bacteria to antibiotics as the 'sensitasome'. Among the identified targets were the essential genes gyrA and gyrB, whose repression increased resistance to multiple antibiotics. 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. Together, these findings establish adaptation-coupled CRISPRi as a simple platform for discovering antibiotic sensitization genes and provide an environmentally safer alternative to conventional antibiotic resistance markers.