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

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.

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Publication Details

Journal
Nucleic Acids Research
Published
2025-04-22
DOI
https://doi.org/10.1093/nar/gkaf353
Citations
3
Primary Topic
CRISPR and Genetic Engineering
Type
article
Field-Weighted Citation Impact
2.42

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article

A CRISPR-SpCas9M-reporting system for efficient and rapid genome editing in Caulobacter crescentus

Yixin Zheng, Hailong Wang, J. Sun, Qingmei Li et al.
3 citations
Nucleic Acids Research
CRISPR and Genetic Engineering
2.42
article

A CRISPR-SpCas9M-reporting system for efficient and rapid genome editing in Caulobacter crescentus

Yixin Zheng, Hailong Wang, J. Sun, Qingmei Li, Wei Tan, Lei Dai, Lianzhong Ai, Mengqing Chen, Yucan Hu, Yingying Li, Xiaoyang Li, Ningning Li, Wei Zhao, Ning Lu, Guoping Zhao, Zhongyue Li, Yujiao Yang, Guiyue Tang, Guohong Zhao, X. R. Lyu, Xin Yu
article en
3 citations

Abstract

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.

Nucleic Acids ResearchVol. 53(8)
Shandong University (CN), Henan University (CN), Shanghai Jiao Tong University (CN), Fudan University (CN), Southern University of Science and Technology (CN), State Key Laboratory of Synthetic Chemistry (CN), Shenzhen Institutes of Advanced Technology (CN), Institute of Microbial Technology (IN), University of Chinese Academy of Sciences (CN), State Key Laboratory of Genetic Engineering
National Natural Science Foundation of China, Chinese Academy of Sciences, National Key Research and Development Program of China, Basic and Applied Basic Research Foundation of Guangdong Province
Zero hunger
Openalex Percentile: Top 12%
CRISPR and Genetic Engineering
2.42
DOIOpen Access PDFOpenAlex
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