Quantitative Analysis of CRISPR-Cas9 Efficiency in Targeted Gene Disruption Across Multiple Bacterial Species

CRISPR-Cas9 genome editing has revolutionized genetic engineering, yet its efficiency varies across bacterial species. This study evaluates the efficiency of CRISPR-Cas9 in disrupting a conserved housekeeping gene across six phylogenetically diverse bacterial species. We hypothesize that editing efficiency is influenced by species-specific factors, necessitating tailored optimization strategies. A comparative experimental design was used to disrupt a conserved gene (e.g., lacZ or rpsL) in Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Clostridium acetobutylicum, Lactobacillus plantarum, and Vibrio cholerae. A plasmid-based CRISPR-Cas9 system with inducible promoters and species-specific sgRNAs was employed. Transformation methods varied by species. Gene disruption was validated using colony PCR, Sanger sequencing, and deep sequencing. Editing efficiency was calculated as the percentage of successfully edited colonies and further quantified using ddPCR and qPCR. Statistical analysis included ANOVA, Tukey’s HSD, and Pearson correlation. Editing efficiencies varied significantly (p < 0.001), ranging from 42.8% (C. acetobutylicum) to 82.3% (E. coli). High GC content negatively correlated with editing efficiency (r = –0.62, p = 0.04). Plasmid size showed a weak, non-significant negative correlation (r = –0.48, p = 0.09). ANOVA and post-hoc tests confirmed significant pairwise differences, particularly between E. coli and C. acetobutylicum (p < 0.001). CRISPR-Cas9 gene disruption efficiency is highly species-dependent, influenced by genomic features, transformation methods, and physiological traits. E. coli and B. subtilis were most amenable, while C. acetobutylicum posed the greatest challenge. Optimization tailored to species-specific biology is essential for effective microbial genome editing.