A CRISPR- Sp Cas9M-reporting system for efficient and rapid genome editing in Caulobacter crescentus

作者：J. Sun, Xin Yu, Guiyue Tang, Mengqing Chen, Yixin Zheng, Yucan Hu, Qingmei Li, Xiaoyang Li, Ningning Li, Zhongyue Li, Yingying Li, Ning Lu, Wei Tan, Yujiao Yang, Xiaoye Lyu, Guohong Zhao, Hailong Wang, Lei Dai, Guo-Ping Zhao, Lianzhong Ai, Wei Zhao · 发表于：Nucleic Acids Research · 年份：2025 · DOI：10.1093/nar/gkaf353 · 被引用次数：5 · 研究领域：CRISPR and Genetic Engineering、Bacterial Genetics and Biotechnology、Microbial Fuel Cells and Bioremediation

As members of the α-proteobacteria group, Caulobacter crescentus and its relatives are wildly studied for their unique asymmetric life cycle and versatile applications in industry, agriculture, and biomedicine. However, genetic manipulation in these bacteria remains challenging, typically requiring time-consuming and labor-intensive procedures. Here, we report a practical CRISPR-SpCas9M-reporting system that overcomes the limitations of SpCas9 expression and CRISPR escape, enabling efficient, markerless, and rapid genome editing in C. crescentus. Two genes encoding for a pair of scaffold proteins were knocked out individually or iteratively, demonstrating their direct involvements in cellular signaling asymmetry. Key components, including the Cas protein, Cas inducer, sgRNA, homologous arms, and reporter, were systematically analyzed and optimized in the system, finally achieving the apparent editing efficiency up to 80% in C. crescentus. Furthermore, we applied the CRISPR-SpCas9M-reporting system to two C. crescentus relatives, Agrobacterium fabrum and Sinorhizobium meliloti, establishing it as an efficient and general editing strategy. We anticipate that this system could be applied to other CRISPR-Cas-recalcitrant organisms, accelerating both basic and applied research in α-proteobacteria.