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

Abstract

Escherichia coli 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 5 and gene 19) 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 E. coli 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 E. coli strains. Protein expression analysis via green fluorescent protein confirms that E. coli 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 E. coli resistance to phages, providing a practical solution for protecting industrial E. coli strains and improving fermentation processes.

1. Introduction

Escherichia coli strains are the most commonly used hosts in industrial production and are widely utilized in the synthesis of various chemicals, including organic acids, alcohols, amino acids, fatty acids, alkanes, etc. Notably, E. coli strains such as MG1655 from the K-12 series and BL21 (DE3) from the B series have become important chassis strains in synthetic biology. However, in actual industrial production, E. coli faces the risk of contamination by bacteriophages. Phages can rapidly kill host bacteria, accumulate at very high concentrations, and spread throughout production facilities. This contamination can lead to a decline in fermentation product quality, reduced production capacity, and economic losses. Therefore, addressing the issue of phage contamination in fermentation processes is urgent.

Various industries have implemented methods to reduce contamination by bacteriophages during fermentation, such as raw material treatment, strain rotation, process changes, plant design adjustments, and extensive cleaning and hygiene practices. However, these measures undoubtedly increase the operating costs of facilities. E. coli possesses several endogenous mechanisms to resist phage infection, including surface receptor mutation, the restriction-modification (R-M) system, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system, the abortive infection system, and the Thoeris system. These mechanisms help E. coli survive phage infection or prevent phage contamination from spreading in the environment. Although E. coli has evolved a wide variety of antiphage strategies through its long-term arms race with bacteriophages, bacteriophages have concurrently evolved various countermeasures to evade these defense systems. Consequently, the endogenous defense mechanisms of E. coli offer limited protection during actual phage infections. Thus, it is necessary to develop more effective measures to defend against bacteriophage infection.