A programmable CRISPR/Cas9-based phage defense system for Escherichia coli BL21(DE3)

Abstract

<jats:title>Abstract</jats:title> <jats:p><jats:italic>Escherichia coli</jats:italic> BL21 is arguably the most popular host for industrial production of proteins, and industrial fermentations are often plagued by phage infections. The CRISPR/Cas system is guided by a gRNA to cleave a specific DNA cassette, which can be developed into a highly efficient programable phage defense system. In this work, we constructed a CRISPR/Cas system targeting multiple positions on the genome of T7 phage and found that it increased the ability of BL21 to defend against phage infection. Furthermore, the targeted loci played a critical role. For better control of expression of CRISPR/Cas9, various modes were tested, and the OD of the optimized strain BL21(pT7cas9, pT7-3gRNA, prfp) after 4 hours of phage infection was significantly improved, reaching 2.0, which was similar to the control culture without phage infection. The viable cell count of the engineered strain in the presence of phage was only one order of magnitude lower than that of the strain with no infection, which further demonstrated the effectiveness of the CRISPR/Cas9 phage defense system. Finally, the engineered BL21 strain under phage attack expressed RFP protein at about 60% the rate of the un-infected control, which was significantly higher than the parent BL21. We successfully constructed a programable CRISPR/Cas9 system to increase the ability of <jats:italic>E. coli</jats:italic> BL21’s to defend against phage infection, and created a resistant protein expression host. This work provides a simple and feasible strategy for protecting industrial <jats:italic>E. coli</jats:italic> strains against phage infection.</jats:p>