Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University, Bielefeld, Germany.
Department of Biotechnology and Food Science, NTNU, Norwegian University of Science and Technology, Trondheim, Norway.
Appl Microbiol Biotechnol. 2020 Jun;104(11):5095-5106. doi: 10.1007/s00253-020-10571-6. Epub 2020 Apr 9.
Gene repression using the endonucleolytically deactivated dCas9 protein and sgRNAs (CRISPR interference or CRISPRi) is a useful approach to study gene functions. Here, we established CRISPRi in Paenibacillus sonchi genomovar Riograndensis SBR5, a plant growth promoting bacterium. CRISPRi system with sgRNAs targeting SBR5 endogenous genes spo0A, yaaT and ydjJ and plasmid-borne gfpUV was constructed and analyzed. Flow cytometry analysis revealed a significant decrease of reporter protein GFPUV signal in P. sonchi strains expressing gfpUV sgRNA in comparison with non-targeting controls. CRISPRi-based repression of chromosomal genes for regulation of sporulation spo0A and yaaT decreased sporulation and increased biofilm formation in SBR5. Repression of the sorbitol catabolic gene ydjJ revealed decreased specific activity of YdjJ in crude cell extracts and reduced biomass formation from sorbitol in growth experiments. Our work on CRISPRi-based gene repression serves as basis for gene function studies of the plant growth promoter P. sonchi SBR5. To our knowledge, the present study presents the first tool for gene repression established in Paenibacillus species.Key points• CRISPRi toward gene repression was applied for the first time in Paenibacillus.• CRISPRi of spo0A and yaaT depleted spores and increased biofilms in SBR5.• CRISPRi-based ydjJ repression decreased specific activity of sorbitol dehydrogenase.
利用内切酶失活的 dCas9 蛋白和 sgRNA 进行基因抑制（CRISPR 干扰或 CRISPRi）是研究基因功能的一种有效方法。在这里，我们在植物促生菌 Paenibacillus sonchi genomovar Riograndensis SBR5 中建立了 CRISPRi。构建并分析了靶向 SBR5 内源性基因 spo0A、yaaT 和 ydjJ 的 sgRNA 和带有质粒 gfpUV 的 CRISPRi 系统。流式细胞术分析显示，与非靶向对照相比，表达 gfpUV sgRNA 的 P. sonchi 菌株中报告蛋白 GFPUV 信号显著降低。CRISPRi 抑制染色体基因 spo0A 和 yaaT 的调控，减少了 SBR5 的孢子形成并增加了生物膜形成。yorJ 基因的蔗糖分解代谢基因 ydjJ 抑制导致粗细胞提取物中 YdjJ 的比活降低，并且在生长实验中从山梨醇形成的生物量减少。我们基于 CRISPRi 的基因抑制工作为植物生长促进剂 P. sonchi SBR5 的基因功能研究提供了基础。据我们所知，本研究首次在芽孢杆菌属中建立了用于基因抑制的 CRISPRi 工具。
