CRISPR interference-based gene repression in the plant growth promoter Paenibacillus sonchi genomovar Riograndensis SBR5

Luciana F Brito,Kerstin Schultenkämper,Volker F Wendisch,Luciane M P Passaglia

doi:10.1007/s00253-020-10571-6

Abstract

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.

Highlights

IntroductionCRISPR (clustered regularly interspaced short palindromic repeats) associated with the type II protein Cas is a wellknown and well-established tool applied for genome editing of diverse organisms
CRISPR associated with the type II protein Cas9 is a wellknown and well-established tool applied for genome editing of diverse organisms
In order to analyze CRISPR interference (CRISPRi) efficiency in P. sonchi SBR5 cells, we chose to express reporter gene gfpUV sgRNA in SBR5 using the plasmid pBV2mp-gfpUV. sgRNA-guided dCas9 activity resulted in a significant decrease of GFPUV medium fluorescence intensity (MFI)

Summary

Introduction

CRISPR (clustered regularly interspaced short palindromic repeats) associated with the type II protein Cas is a wellknown and well-established tool applied for genome editing of diverse organisms. CRISPR interference (CRISPRi) has been applied for gene repression (Xu and Qi 2019). CRISPRi-based gene repression has been applied in bacteria for many purposes, for instance, to redirect metabolic fluxes toward industrially relevant products (Cleto et al 2016; Kim et al 2017; Schultenkämper et al 2020). Multiplexed CRISPRi was established in Bacillus licheniformis by targeting genes involved in byproduct synthesis and L-valine degradation pathway, resulting in up to 80% increase in L-valine titers (Zhan et al 2020). In Bacillus subtilis, CRISPRi-mediated repression of 16 genes on the branch metabolic pathways of amino acid biosynthesis resulted in up to 0.75 g L−1 surfactin production (Wang et al 2019). CRISPRi was established in the methylotrophic Bacillus methanolicus, controlling its sporulation process by repression of sporulation regulator Spo0A and controlling its mannitol catabolism by CRISPRi of mannitol-1-phosphate dehydrogenase mtlD gene (Schultenkämper et al 2019)

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