Escherichia coli 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 the system increased the BL21’s defense ability against phage infection. Furthermore, the targeted loci on phage genome 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 h of phage infection was significantly improved, reaching 2.0, which was similar to the control culture without phage infection. Although at later time points, the defensive ability of CRISPR/Cas9 systems were not as obvious as that at early time points. 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% of the un-infected control, which was significantly higher than the parent BL21. In this work, we successfully constructed a programable CRISPR/Cas9 system to increase the ability of E. coli 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 E. coli strains against phage infection.

中文翻译：

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一种基于CRISPR / Cas9的可编程噬菌体防御系统，用于大肠杆菌BL21（DE3）。

大肠杆菌BL21可以说是蛋白质工业化生产中最受欢迎的宿主，并且工业发酵常常受到噬菌体感染的困扰。CRISPR / Cas系统在gRNA的指导下裂解特定的DNA盒，可将其开发为高效的可编程噬菌体防御系统。在这项工作中，我们构建了针对T7噬菌体基因组上多个位置的CRISPR / Cas系统，发现该系统增强了BL21对噬菌体感染的防御能力。此外，噬菌体基因组上的靶向基因座发挥了关键作用。为了更好地控制CRISPR / Cas9的表达，测试了多种模式，噬菌体感染4 h后优化菌株BL21（pT7cas9，pT7-3gRNA，prfp）的吸光度得到明显改善，达到2.0，这类似于没有噬菌体感染的对照培养物。尽管在较晚的时间点，CRISPR / Cas9系统的防御能力不如较早的时间点明显。存在噬菌体的工程菌株的活细胞计数仅比没有感染的菌株低1个数量级，这进一步证明了CRISPR / Cas9噬菌体防御系统的有效性。最后，经噬菌体攻击的工程BL21菌株在未感染对照中表达RFP蛋白的比例约为60％，明显高于亲本BL21。在这项工作中，我们成功构建了一个可编程的CRISPR / Cas9系统，以增强大肠杆菌BL21抵抗噬菌体感染的能力，并创建了一个抗性蛋白表达宿主。