Optimized CRISPR\nInterference\nSystem for Investigating Pseudomonas alloputida Genes Involved in Rhizosphere Microbiome Assembly

Pseudomonas alloputida KT2440 (formerly P. putida) has become\nboth a well-known chassis organism\nfor synthetic biology and a model organism for rhizosphere colonization.\nHere, we describe a CRISPR interference (CRISPRi) system in KT2440\nfor exploring microbe–microbe interactions in the rhizosphere\nand for use in industrial systems. Our CRISPRi system features three\ndifferent promoter systems (XylS/<i>P</i><sub>m</sub>, LacI/<i>P</i><sub>lac</sub>, and AraC/<i>P</i><sub>BAD</sub>) and a dCas9 codon-optimized for Pseudomonads, all located on a\nmini-Tn7-based transposon that inserts into a neutral site in the\ngenome. It also includes a suite of pSEVA-derived sgRNA expression\nvectors, where the expression is driven by synthetic promoters varying\nin strength. We compare the three promoter systems in terms of how\nwell they can precisely modulate gene expression, and we discuss the\nimpact of environmental factors, such as media choice, on the success\nof CRISPRi. We demonstrate that CRISPRi is functional in bacteria\ncolonizing the rhizosphere, with repression of essential genes leading\nto a 10–100-fold reduction in P. alloputida cells per root. Finally, we show that CRISPRi can be used to modulate\nmicrobe–microbe interactions. When the gene <i>pvdH</i> is repressed and P. alloputida is\nunable to produce pyoverdine, it loses its ability to inhibit other\nmicrobes <i>in vitro.</i> Moreover, our design is amendable\nfor future CRISPRi-seq studies and in multispecies microbial communities,\nwith the different promoter systems providing a means to control the\nlevel of gene expression in many different environments.