State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Fujian, 361102, China.
Microb Biotechnol. 2021 Nov;14(6):2700-2710. doi: 10.1111/1751-7915.13805. Epub 2021 Mar 27.
Purple non-sulfur photosynthetic bacteria (PNSB) such as Rhodobacter capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for genome editing and transcriptional regulation in R. capsulatus. Template-free disruption method mediated by CRISPR/Cas12a reached ˜ 90% editing efficiency when targeting ccoO or nifH gene. When both genes were simultaneously edited, the multiplex editing efficiency reached > 63%. In addition, CRISPR interference (CRISPRi) using deactivated Cas12a was also evaluated using reporter genes egfp and lacZ, and the transcriptional repression efficiency reached ˜ 80%. In summary, our work represents the first report to develop CRISPR/Cas12a-mediated genome editing and transcriptional regulation in R. capsulatus, which would greatly accelerate PNSB-related researches.
紫色非硫光合细菌（PNSB）如荚膜红细菌可用作基础研究和各种生物技术应用的多功能平台。在本研究中，我们试图从弗朗西斯氏菌属中开发出 II 类 RNA 指导的 CRISPR/Cas12a 系统，用于荚膜红细菌的基因组编辑和转录调控。当靶向 ccoO 或 nifH 基因时，由 CRISPR/Cas12a 介导的无模板破坏方法达到了约 90%的编辑效率。当同时编辑两个基因时，多重编辑效率达到>63%。此外，还使用失活 Cas12a 的 CRISPR 干扰（CRISPRi）评估了报告基因 egfp 和 lacZ 的转录抑制效率，转录抑制效率达到约 80%。总之，我们的工作首次报道了在荚膜红细菌中开发 CRISPR/Cas12a 介导的基因组编辑和转录调控，这将极大地加速 PNSB 相关研究。
