Regulatory Time-Kill Sequencing Maps Antibiotic-Specific Transcriptional Control of Escherichia coli Persistence at Genome Scale

Despite its importance, a systems-level, quantitative understanding of how transcriptional regulation shapes persistence kinetics has remained limited, largely because conventional time-kill assays are inherently low-throughput.

Here, we develop Regulatory Time-Kill Sequencing (RTK-seq), a high-throughput functional genomics strategy that combines a pooled CRISPR interference (CRISPRi) library targeting transcription factors with time-resolved antibiotic selection and deep barcode sequencing. RTK-seq simultaneously resolves multiphasic killing trajectories for 174 Escherichia coli transcriptional regulators over five orders of magnitude in survival.