• D-CAPS, a double-site CRISPR-Cas9 system, provides complete resistance against T7 phage in E. coli, with no plaques even at high MOI.
• D-CAPS maintains normal growth and protein expression in E. coli, comparable to wild-type strains, even under phage challenge.
• The system is versatile, effective across different E. coli strains including MG1655 and BL21(DE3), highlighting its industrial applicability.
• D-CAPS offers a robust solution to phage contamination in fermentation, potentially reducing economic losses and improving product quality.