D-CAPS: an efficient CRISPR-Cas9-based phage defense system for <italic>E</italic>. <italic>coli</italic>

<i>Escherichia coli</i> is widely used in industrial chemical synthesis but faces significant challenges due to bacteriophage contamination, which reduces product quality and yield. Therefore, developing an efficient antiphage system is essential. In this study, we develop a CRISPR-Cas9-based antiphage system (CAPS) targeting essential genes of the T7 phage (gene <i>5</i> and gene <i>19</i>) with single gRNAs transformed into MG1655 strains expressing Cas9. While CAPS provides limited resistance, with plating efficiencies ranging from 10 ^-5 to 10 ^-1, further optimization is needed. To enhance efficacy, we design a double-site-targeting CRISPR-Cas9-based antiphage system (D-CAPS). D-CAPS demonstrates complete resistance, with no plaques observed even at a high multiplicity of infection (MOI of 2), and growth curve analysis reveals that antiphage <i>E</i>. <i>coli</i> strains grow normally, similar to the wild-type strain, even at a high multiplicity of infection. Furthermore, D-CAPS is effective against BL21(DE3) strains, showing strong resistance and demonstrating its versatility across different <i>E</i>. <i>coli</i> strains. Protein expression analysis via green fluorescent protein confirms that <i>E</i>. <i>coli</i> carrying D-CAPS could maintain normal protein expression levels even in the presence of phages, comparable to wild-type strains. Overall, D-CAPS offers a robust and versatile approach to enhancing <i>E</i>. <i>coli</i> resistance to phages, providing a practical solution for protecting industrial <i>E</i>. <i>coli</i> strains and improving fermentation processes.