Genetic knockout of pagP in avian pathogenic Escherichia coli using CRISPR-Cas9/λ-Red system and growth characterization

Avian pathogenic Escherichia coli ( APEC ) causes costly colibacillosis across poultry systems, underscoring the need for safe, targeted vaccine antigens. The outer-membrane enzyme pagP, which palmitoylates lipid A and aids innate-immune evasion, is a promising subunit vaccine candidate if it proves non-essential for baseline fitness. We developed and documented a reproducible CRISPR-Cas9/λ-Red workflow to delete pagP in field-derived APEC strain MS1657, constructed donor DNA with short homologous arms, and verified editing by PCR/Sanger sequencing. Editing was efficient (4/5 colonies positive), and whole-genome sequencing of wild type strain and isogenic MS1657Δ pagP line confirmed a precise locus deletion with no additional coding changes or structural alterations, establishing genome-wide specificity. Following a multi-step counter-selection process, editing plasmid-free Δ pagP clones were obtained. To assess fitness, we compared growth kinetics in LB at 37°C using nine biological replicates per strain with OD₆₀₀ readings every 5 min for 24 h. Doubling times did not differ (MS1657: 59.4 ± 5.4 min; MS1657Δ pagP : 60.8 ± 9.9 min; two-tailed t-test, P = 0.697), indicating that pagP is dispensable under nutrient-rich conditions. These data, together with the protein’s surface exposure and functional relevance, support pagP as a safe, non-essential, and tractable antigen for subunit vaccine development. This study provides a stepwise, genome validated knockout pipeline for APEC that can be applied to additional loci and related poultry pathogens. Future embryo and chick challenge studies will define the contributions of pagP to virulence, host recognition, and protection, advancing rational antigen selection for colibacillosis control.