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

As members of the α-proteobacteria group, Caulobacter crescentus and its relatives are known for their asymmetric life cycle and comprehensive applications in gene delivery, agricultural biotechnology, and the production of high-value compounds. However, genetic manipulations of these bacteria are often time-consuming and labor-intensive due to the lack of efficient genome editing tools. Here, we report a practical CRISPR/ Sp Cas9M-reporting system that overcomes the limitations of Sp Cas9 expression, enabling efficient, markerless, and rapid genome editing in C. crescentus . As a demonstration, we successfully knocked out two genes encoding the scaffold proteins, achieving apparent editing efficiencies up to 80%. Key components, including the Cas protein, Cas inducer, sgRNA, homologous arms, and reporter, were systematically analyzed and optimized to enhance the editing efficiency or decrease the cell lethality. A nearly zero off-target ratio was observed after the curing of the editor plasmid in editing strains. Furthermore, we applied the CRISPR/ Sp Cas9M-reporting system to two C. crescentus relatives, Agrobacterium fabrum and Sinorhizobium meliloti , establishing it as an efficient and reliable editing strategy. We anticipate that this system could be applied to other hard-to-edit organisms, accelerating both basic and applied research in α-proteobacteria.